Critical Reviews in Oncology/Hematology 38 (2001) 139– 170

www.elsevier.com/locate/critrevonc

Review
Mechanisms of action of flavopiridol
H.H. Sedlacek *
A6entis Pharma Deutschland GmbH, Central Biotechnology, P.O. Box 1140, 35001 Marburg, Germany

Received 28 February 2000; received in revised form 30 September 2000; accepted 23 October 2000

Contents
1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

2. Inhibition of kinases in relation to intracellular concentration of flavopiridol . . . . . . . . . . 141

3. Function and inhibition of cyclin dependent kinases . . . . . . . . . . . . . . . . . . . . . . . . 142

3.1. *Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
3.2. Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

4. Characteristics of the antiproliferative activity of flavopiridol . . . . . . . . . . . . . . . . . . . 145

4.1. Induction of apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
4.2. Interaction with cytostatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
4.3. Activity in resting cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
4.4. Activity in relation to pRb expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
4.5. Activity in relation to p53 expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
4.6. Activity in relation to Bcl-2 expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

5. Inhibition of other kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

5.1. Interaction with receptors for the EGF family . . . . . . . . . . . . . . . . . . . . . . . . . 154
5.2. Inhibition of receptor associated protein kinases . . . . . . . . . . . . . . . . . . . . . . . . 155
5.3. Inhibition of protein kinase C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

6. Modulation of multidrug resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

7. Antiangiogenic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Biography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

* Tel.: +49-6421-392768; fax: + 49-6421-394663.

E-mail address: hans-harald.sedlacek@aventis.com (H.H. Sedlacek).

1040-8428/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
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140 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170

Abstract

Flavopiridol inhibits phosphokinases. Its activity is strongest on cyclin dependent kinases (cdk-1, -2, -4, -6, -7) and less on
receptor tyrosine kinases (EGFR), receptor associates tyrosine kinases (pp60 Src) and on signal transducing kinases (PKC and
Erk-1). Although the inhibiting activity of flavopiridol is strongest for cdk, the cytotoxic activity of flavopiridol is not limited to
cycling cells. Resting cells are also killed. This fact suggests that inhibition of cdks involved in the control of cell cycle is not the
only mechanism of action. Inhibition of cdk’s with additional functions (i.e. involved in the control of transcription or function
of proteins that do not control cell cycle) may contribute to the antitumoral effect. Moreover, direct and indirect inhibition of
receptor activation (EGFR) and/or a direct inhibition of kinases (pp60 Src, PKC, Erk-1) involved in the signal transduction
pathway could play a role in the antiproliferative activity of flavopiridol. From pharmacokinetic data in patients it can be
concluded that the inhibitory activity (IC50) of flavopiridol on these kinases is in the range of concentrations that might be
achieved intracellularly after systemic application of non-toxic doses of flavopiridol. However, no in situ data from flavopiridol
treated cells have been published yet that prove that by inhibition of EGFR, pp60 Src, PKC and/or Erk-1 (in addition to
inhibition of cdk’s) flavopiridol is able to induce apoptosis. Thus many questions regarding the detailed mechanism of antitumoral
action of flavopiridol are still open. For the design of protocols for future clinical studies this review covers the essential
information available on the mechanism of antitumoral activity of flavopiridol. The characteristics of this antitumoral activity
include: High rate of apoptosis, especially in leukemic cells; synergy with the antitumoral activity of many cytostatics;
independence of its efficacy on pRb, p53 and Bcl-2 expression; lack of interference with the most frequent multidrug resistance
proteins (P-glycoprotein and MRP-190); and a strong antiangiogenic activity. Based on these pharmacological data it can be
concluded that flavopiridol could be therapeutically active in tumor patients: independent on the genetic status of their tumors or
leukemias (i.e. mutations of the pRb and/or p53, amplification of bcl-2 ); in spite of drug resistance of their tumors induced by first
line treatment (and caused by enhanced expression of multidrug resistance proteins); in combination with conventional
chemotherapeutics preferentially given prior to flavopiridol; and due to a complex mechanism involving cytotoxicity on cycling
and on resting tumor cells, apoptosis and antiangiogenic activity. In consequence, flavopiridol is a highly attractive, new
antitumoral compound and deserves further elucidation of its clinical potency. © 2001 Elsevier Science Ireland Ltd. All rights
reserved.

Keywords: cdk; Phospokinases; Inhibitors; Flavopiridol; Mechanism of action; Apoptosis; Cell cycle; Multidrug resistance; Antiangiogenic activity

1. Introduction cdk7. The broadness of its activity on the different

The new compound flavopiridol (L868 275,
NSC649 890, HMR1275) was selected for further stud-
ies [1,2] because of:
1. Its high potency to inhibit the proliferation of a
broad range of human tumor cell lines after pro-
longed exposure time
2. Its potency to inhibit tyrosine kinases and serine
kinases [1,3]
3. The discrepancies between its high degree of cytoxi-
city and
4. Its potency to inhibit known kinases as well as the
lack of correlation between its cytoxicity and the
sensitivity of the respective test cells to growth fac-
tors and
5. Its potency to inhibit in vivo a broad type range of
human tumors, leukemias and lymphomas [1,2,4 –8].
In view of its strong antitumoral activity the question
of the mechanism of action of flavopiridol was first
taken up in 1991 [2,9]. It was speculated [1] that inhibi-
tion of both types of kinases, i.e. tyrosine as well as
serine phosphokinases may synergistically contribute to
the strong antiproliferative potency of flavopiridol.
In addition it was found that flavopiridol inhibits the
cell cycle and is a strong inhibitor of cyclin dependent
kinases (cdk’s), including cdk1, cdk2, cdk4, cdk6 and
cdk’s and the strengths of its activity on cdk-4 has been
regarded as unique [2].
The essential role of cdk’s in driving cells through the
cell cycle [10] implies that flavopiridol should act on
cycling cells via inhibition of cdk’s. Several groups
could confirm this assumption, others, however, found
that flavopiridol is also cytotoxic for resting cells.
To explain this discrepancy many experiments were
performed in order to elucidate the mechanism of ac-
tion of flavopiridol. Tests were thus conducted to estab-
lish whether the cytotoxic activity of flavopiridol is
dependent on pRb expression or on the expression of
other proteins being involved in cell survival, i.e. p53 or
Bcl-2 or on the cell line being investigated, its func-
tional state or any other experimental condition.
The overall results of these experiments indicate that
the mechanism of action of flavopiridol seems to be
more complex than predicted and to depend on various
experimental conditions including the type of the target
cells chosen.
Clinical studies with flavopiridol have already been
started and clinical tumor responses could be observed
in most of the phase I [11 –13] and phase II studies [14]
on different types of progressive tumors refractory to
conventional treatment.
 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170 141

However, another clinical phase II study failed to
show any effect of flavopiridol on metastatic renal
carcinoma [15].
The reason for this discrepancy is unknown yet, but
it could be that the dose and treatment schedule applied
may not have been appropriate in view of the complex
mechanisms of action of flavopiridol. Thus the aim of
this review is to report in a critical manner all the
various suggested mechanisms so that the reader may
be able to design further clinical studies by taking the
complexity of the mechanisms of action of flavopiridol
into account.
2. Inhibition of kinases in relation to intracellular
concentration of flavopiridol
Mammalian protein kinases include receptor kinases,
receptor associated protein kinases, signal transducing
kinases and cyclin dependent kinases [16].
Flavopiridol has a proven inhibiting activity on a
variety of such cellular protein kinases (see Table 1) [2].
The potency of this inhibiting activity, however, differs
among the different types of kinases. Moreover, the
inhibition of which kinase at which concentration is
relevant for the antitumoral activity in vitro and in
vivo, particularly in patients, is unknown. One way to
answer this question is to estimate the concentration of
flavopiridol in the extravascular compartments after
systemic application. As flavopiridol is not only soluble
in water but also slightly to sparingly soluble in etha-
nol, n-octanol or methanol [2], it can be expected that
flavopiridol is able to penetrate cell membranes and to
bind to intracellular and extracellular lipids and
lipoproteins.
Pharmacokinetic studies in mice and rats [2] showed
that after i.v. injection plasma levels of flavopiridol
decline quickly (T1/2a of 16 min (mice) and 10.5 min
(rats)). However, the elimination of flavopiridol from
the blood plasma was prolonged (T1/2b of 201 min
(mice) and 112 min (rats)) and the volume of distribu-
tion at steady state (Vdss) was relatively high (8.3 l/kg
(rats)) [2]. The short T1/2a and the high Vdss value in
rodents suggest a high distribution of the drug into
extravascular compartments [17].
Clinical phase I studies in tumor patients, however,
showed non-homogenous pharmacokinetic results.
Most ( : 70%) patients exhibited a course of blood
plasma elimination of flavopiridol similar to the one
found in mice and rats with a relatively long half-life
T1/2b of 11.6 (range 1.3 –29.1 h) and a high Vdss of
131.16 l/m2. However, some (approximately about one
third) of patients showed aberrant pharmacokinetic
profiles that included postinfusion single or saw tooth
transient elevations of flavopiridol blood plasma con-
centrations [12].
The median maximal steady state plasma flavopiridol
concentration (Cpss) in patients was 0.27 (range 0.17 –
2.94) mM (after the administration of the maximal
tolerated dose of 50 mg/m2/dx3) and 0.55 (range 0.36 –
4.21) mM (after infusion of the toxic dose low (TDL:
122.5 mg/m2/dx3) administered together with antidi-
arrheal prophylaxis (Loperamide) and causing hypoten-
sion as the dose limiting toxicity) [12,13,18].
In another phase I study the mean Cpss after the
administration of 56 mg/m2/dx3 was 0.469 0.16 mM
[19].

Table 1
Activity of Flavopiridol on cellular kinases

Family Kinase IC50 (mM) Ref.

50.3 B40 ]40

EGF receptor family EGF receptor (TPK) 21–25 Sedlacek et al. [1]
Losiewicz et al. [81]
Receptor associated src family kinases pp60 Src 34 Czech et al. [3]
In T-Ly Lck 50 Czech et al. [3]
In Mo/PMN Hck 500 Czech et al. [3]
In T/B-Ly Fyn 500 Czech et al. [3]
Signal transducing kinases PKA 122–145 Sedlacek et al. [1]
PKC 6 Losiewicz et al. [81]
Czech et al. [5]
Erk-1 16 Webster et al. [320]
Cyclin dependent kinases (direct inhibition) cdk1/cyclin B 0.03–0.4 Losiewicz et al. [81]
cdk2/cyclin A 0.1 Carlson et al. [86]
cdk2/cyclin E 0.1 Carlson et al. [87]
cdk4/cyclin D 0.02–0.04 Carlson et al. [86]
cdk6/cyclin D 0.06 Singh et al. [88]
cdk7/cyclin H 0.11–0.3 Carlson et al. [36]
142 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170
The high distribution volume of flavopiridol at steady
state (Vdss) in rats and patients indicates that the distri-
bution of flavopiridol leads to a higher concentration of
flavopiridol in the extravascular compartments com-
pared to blood plasma. As the concentration in blood
plasma of flavopiridol 12 h after infusion into patients
was very similar to the concentration found in their
pleura or pericardial fluids (dose 16 mg/m2/x3: 59 nmol/l
in plasma; 57 nmol/l in pleura; dose 122.5 mg/m2/x3: 528
nmol/l in plasma; 409 nmol/l in pleura) [12] it can be
concluded that the concentration of flavopiridol in the
interstitial (i.e. extravascular and extracellular) compart-
ments is similar to that found in the plasma. In conse-
quence, it can be assumed that the high Vdss is mainly
caused by an easy cell membrane passage possibly
leading to an enrichment of flavopiridol in the intracel-
lular compartment. However, no data on intracellular
concentrations of flavopiridol are available yet. Indeed,
such data respresent a cornerstone in the discussion
concerning potentially relevant mechanisms of action of
flavopiridol and should be performed as early as possible.
As the intracellular concentration of flavopiridol is
unknown the area under the blood plasma concentration
curve (AUC) is taken as a surrogate parameter. The
AUC should reflect the amount of flavopiridol ex-
changed between the extravascular compartments and
blood over time. The AUC for flavopiridol varied
considerably between the individual patients and dosage
groups and was in the range between 16.8 mM h (after
infusion of 50 mg/m2/dx3), 38.3 mM h (after infusion of
78 mg/m2/dx3; this dose is the MTD of flavopiridol plus
antidiarrheal prophylaxis) and 144 mM h (after infusion
of the TDL of 122.5 mg/m2/dx3, i.e. the highest dose that
was administered) [12].
Taking these pharmacokinetic data into consideration
the following concentrations can be chosen as thresholds
to decide whether the in vitro data of flavopiridol may
have some relevance for antitumoral activity in vivo.
Concentrations that are achievable intracellularly in
vivo:
With 50.3 mM Mean Cpss in patients af-
certainty ter administration of the
MTD (50–56 mg/m2/dx3)
With some 55.0 mM Mean Cpss in patients af-
probability ter administration of the
TDL under antidiarrheal
therapy (122.5 mg/m2/
dx3)
With low 540 mM Derived from the AUC
probability in patients after adminis-
tration of the MTD un-
der antidiarrheal therapy
(78 mg/m2/dx3)
Probably not \40 mM
Using these parameters it can be concluded that
flavopiridol might be active in vivo via inhibition of
Cdk’s, PKC, EGFR, pp60 Src and Erk-1 but probably
not via inhibition of Lck, Hck, Fyn and PKA (see Table
1).
3. Function and inhibition of cyclin dependent kinases
3.1. Function
Cyclin dependent kinases consist of a catalytic subunit
(cdk) and a regulatory subunit (cyclin). More than eight
human cdk’s have been described so far. The most
important ones involved in the cell division cycle are:
cdk1 (=cdc2), cdk2, cdk4, cdk5, cdk6, cdk7 and cdk8.
All of these cdk’s are regulated by the transient associ-
ation with one member of the cyclin family including
cyclin A, cyclin B1 –B3, cyclin D1 –D3, cyclin E, cyclin
H and cyclin C. Other cdk1-related kinases have been
sequenced and await identification of their regulatory
partners and cell cycle regulatory functions [10,20].
Progression through each step of the cell cycle is
thought to be regulated by cdk-complexes. The impor-
tant ones are:
cdk4/cyclin D (1–3), Mid G1
cdk6/cyclin D (1–3)
cdk2/cyclin E Late G1
phase
cdk2/cyclin E, cdk2/cyclin A G1/S
transition
cdk2/cyclin A S-phase
cdk1/cyclin B, cdk1/cyclin A G2/M
transition
Cyclin-dependent kinases are for their part regulated
through ‘check-point control’ at defined restriction
points that ensure that each step of the cell cycle is
completed before the cell can proceed through the next
step [21,22].
The mechanisms regulating cyclin-dependent kinases
[10,20,23 –29] include:
1. Post-translational modifications by activating en-
zymes (cdc25C phosphatase) and inhibitory enzymes
(wee1 kinase);
2. Formation of cyclin complexes;
3. Association with inhibitory subunits (e.g. p16INK4A,
p15INK4B, p21Cip1, p27Kip1, p18 (for cdk4, -2, and
-1)), some of them (p16, p15, p18) inhibiting by
displacing the cyclin subunit.
 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170
Fig. 1. Inhibition of cdk’s and cell cycle progression.
4. Association with regulatory subunits of unknown,
yet essential functions (p9CDShs, p15cdkBP).
5. Ubiquitin-dependent degradation of the cyclin
subunit;
6. Intracellular translocations and association with
subcellular structures (centrosomes, microtubules).
The regulatory sites of cyclin-dependent kinases in-
clude the ATP-binding pocket in the catalytic subunit
of the cdk/cyclin complexes that contain two amino
acids that inhibit kinase activity when phosphorylated
[30 – 32]. The protein substrate binding site is located in
the cleft between the two lobes of the catalytic subunit.
Its phosphorylation by cdk7/cyclin H appears to be
essential for kinase activity [33 – 36].
In addition cdk7/cyclin H complexes are also inti-
mately associated with the transcriptional machinery:
specifically, they are found within the basal transcrip-
tion factor TFIIH in mammalian cells. Cdk7/cyclin H is
constitutively active and capable of phosphorylating the
carboxy-terminal domain of RNA polymerase II [37].
Cyclin D molecules have redundant cell functions,
however, their tissue expression and regulation are very
different: cyclin D1 is more prevalent in epithelial tis-
sues whereas cyclins D2 and D3 are more prevalent in
hematopoietic tissues [38].
The expression of cyclin D1 in normal cells varies
with the phase of the cell cycle with a peak in early G1
followed by a decline during S- and G2-M phase. The
expression of cyclin D is regulated by transcriptional
and translational mechanisms [39]. In contrast, the
expression of most of the other cyclins (i.e. cyclin B1,
A, E) is regulated by degradation processes [40 –44].
The excessive, aberrant or inappropriate expression
of cdk’s and/or cyclins and their regulatory proteins
143
during cell cycle seems to have an essential role in the
pathogenesis of diverse tumor diseases. Cyclins D1, D2,
A and E have been reported to be overexpressed in
many different types of cancer [45 –50] and to be corre-
lated directly and quantitatively with tumor aggressive-
ness and invasiveness [47,51]. Cdk1, cdk4 or cdk7 have
been found to be overexpressed or mutated in several
tumors [46,49,52,53]. Likewise p16 or p15 were found
to be deleted, mutated or aberrantly methylated in
many tumor types, both in vitro and in vivo [49,54,55].
Main cellular substrates for cyclin dependent kinases
are proteins of the retinoblastoma protein family, i.e.
pRb, p107 and p130 (see Fig. 1). In the hypoposphory-
lated state all three so-called pocket proteins are capa-
ble of inhibiting growth in certain cell types by forming
complexes with and inhibiting a number of partner
molecules (see Fig. 1). The functional inactivation of
pocket proteins takes place by hyperphosphorylation
[56 –61]. Loss-of-function mutations of pocket proteins,
i.e. of pRb are very common in small cell lung cancer
[62] and other tumor types [63 –66], indicating their
function as tumor suppressors [58,67 –74].
pRb and p130 are detected in resting cells as low
phosphorylated forms. When cells progress through the
G0/G1 transition and through mid G1, p130 is hyper-
phosphorylated and levels of hyperphosphorylated
p130 are downregulated as cells move to S phase
[58,67 –74]. In constrast very little-to-no p107 is de-
tected in quiescent cells. However, p107 accumulates
significantly as cells progress through the cell cycle
[67,75,76].
Cdk’s phosphorylate multiple sites (primarily serine
threonine sites) in pRb and most likely in p107 and
p130 [76]. Phosphorylation of pRb, p107 and p130 is
144 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170
associated with the loss of their growth suppressive
activities. Such activities are mediated through the asso-
ciation of pocket proteins with a number of cellular
proteins including the members of the E2F family
(E2F-1, -2, -3, -4, -5) of transcription factors and other
transcription factors involved in cell proliferation (i.e.
c-Jun, ATF-2, Sp1, AhR, TF II D, c-Myc, N-Myc),
transcription factors controlling expression of RNA
polymerases (TFIIIB, UBF)), tissue specific transcrip-
tion factors involved in the differentiation of myocytes,
adipocytes and epithelial cells, cdk-subunits (cyclin
D1 – 3, cyclin A, E) and inhibitors of the tumor sup-
pressor p53 (MDM-2) [76].
For phosphorylation D-type cyclin/cdk complexes
interact with all three pocket proteins [45,77,78], while
cyclin E and cyclin A/cdk2 complexes form stable
complexes with p107 and p130, but not with pRb.
For inactivation of the growth suppressor activity of
pRb, phosphorylation of its Ser-795 by cdk4/cyclin D1
complexes is essential [79].
Cdk also phosphorylate E2F. Phosphorylation by
cdk4/cyclin (D1 –3) in the G1 phase prevents interac-
tion of E2F with and inhibition by pocket proteins
whereas hyperphosphorylation of E2F by cdk2/cyclin A
complexes in the late S-phase leads to its inactivation
[21,22].
3.2. Inhibition
Activity of flavopiridol is strongest on cdk’s (see
Table 1). Flavopiridol directly inhibits purified acti-
vated cdk1/cyclin B complexes from sea star and to the
same extent human cdk1.
This could be proven by experiments in which the
kinase activity of cdk1 antibody immunoprecipitates
(obtained (at 9–12 h) after release of breast carcinoma
cells from aphidicolin block of DNA polymerase [8]
into drug-free medium when the cells are in late G2 or
passing into M-phase) was potently inhibited by
flavopiridol. Neither autophosphorylation of cdk1 ki-
nase nor any ATPase activity was detected [9,80 –82].
Inhibition of cdk1 by flavopiridol could be reduced by
an excessive concentration of ATP [81].
Analysis of cdk1 kinase activity in cells synchronized
at the G1/S transition by aphidicolin and treated with
flavopiridol revealed inhibition of cdk1 kinase activity
relative to control activity at every time in the S-, G2-
or M-phase and concomitantly retardation in cell cycle
progression through S-phase and accumulation in G2
[83].
Cdk1 immunoprecipitates isolated from such syn-
chronized and flavopiridol-treated cells either by a C-
terminal specific or by a PSTAIRE motif specific
antiserum showed a decrease in [32P]-orthophosphate
labeling, which indicates that the activity of those ki-
nases phosphorylating cdk1 (i.e. cdk7/cyclin H) is also
inhibited by flavopiridol [83].
This result is in marked contrast to other growth
inhibitory agents such as nitrogen mustard, methotrex-
ate, camptothecin or etoposide that are known to in-
duce a preserved or increased tyrosine phosphorylation
of cdk1 [84].
Control experiments including Western blot on cdk1
immunoprecipitates showed that the relative mass of
cdk1 was not altered during the time of flavopiridol
exposure (in contrast to cdk1 protein reduction after
exposure to protein synthesis inhibitors emetine and
cycloheximide) during which phosphorylation and cdk1
kinase activity were decreased. Furthermore, high doses
of flavopiridol did not inhibit [32S]-methionine incorpo-
ration into immunoprecipitated cdk1 or into proteins in
whole cell lysates [83].
In addition, no decrease of cyclin B was detected by
an anti-cyclin B antibody in cdk1 immunoprecipitates
after flavopiridol treatment of aphidocolin synchro-
nized cells [83]. Thus it may be concluded that flavopiri-
dol inhibits phosphorylation and kinase activity of
cdk1/cyclin B but does not alter the protein level of
either protein by a specific or a global effect on protein
synthesis.
Using breast carcinoma cells (the same as used for
the cdk1 experiments) synchronized through block in
the M-phase by nocodazole [85 –87] and using specific
antisera to cdk2 it was revealed that cdk2 was likewise
directly inhibited by flavopiridol. Phosphoamino acid
analysis showed both threonine and tyrosine phospho-
rylation of cdk2 to be abrogated in the presence of
flavopiridol [82,86,87]. Thus, it could be concluded that
flavopiridol inhibits the normal phosphorylation and
activity of cdk2 and by this mechanism prevents S-
phase entry of cycling cells (see Fig. 1).
Moreover, using immune complex kinase assays (sim-
ilar to cdk1 and cdk2) for cdk4 and cdk7, flavopiridol
was shown to inhibit both enzymes very efficiently (see
Table 1) [86,87].
In tumor cells known to lack cdk4, flavopiridol in-
hibited the activity of cdk6 [88] in exponentially grow-
ing cells. Such cdk4 deficient cells synchronized at
G0/G1 by serum starvation were unable to progress to
S phase after serum stimulation when flavopiridol was
present. This G1 arrest also coincided with dephospho-
rylation of the cdk6 (and cdk4) specific phosphoryla-
tion site of pRb, detected by a specific antibody [88].
Altogether, it can be concluded that flavopiridol can
inhibit cell cycle progression by a variety of mecha-
nisms related to cdk inactivation (see Fig. 1) including:
1. Inhibition of cdk4/cyclin D and cdk6/cyclin D lead-
ing to
(a) Inhibition of phosphorylation of pRb, p107
and p130, which induces binding to their part-
ner molecules and results in inhibition of their
transcriptional activity (see Fig. 1)
(b) Inhibition of activation of E2F and
 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170
(c) Stop in G1 progression
2. Inhibition of cdk2/cyclin E and cdk2/cyclin A lead-
ing to
(a) Inhibition of further phosphorylation and inac-
tivation of pRb, p107 and p130
(b) Inhibition of inactivation of E2F (by cdk2/cy-
clin A) and
(c) Retardation in cell cycle progression through
the S-phase and accumulation in G1 and G2
3. Inhibition of cdk1/cyclin A and cdk1/cyclin B lead-
ing to
(a) Inhibition of activation of topoisomerase acti-
vators, of lamin proteoglycans histone 1
protein and chromatin condensation and
(b) Retardation in cell cycle progression through
S-phase and accumulation in G2 phase
4. Inhibition of cdk7/cyclin H complexes leading to
(a) Reduced activation of other cdk’s (i.e. cdk4/cy-
clin D, cdk1/cyclin B)
(b) Reduced activation of RNA polymerase
In addition to cdk7 some other cdk’s, i.e. cdk8 and
cdk9 have been proposed to participate directly in the
control of transcription and activation of RNA-poly-
merase [89,90]. Inhibition of such cdk’s by flavopiridol
may directly influence expression of cyclin D1 and D3
[91].
To understand how flavopiridol binds to and inhibits
cyclin dependent kinases the X-ray structure of cdk2 in
complex with the deschlorophenyl derivative L868 276
of flavopiridol was determined and compared with the
X-ray structure of the cdk2-ATP-complex [92].
The deschlorophenyl flavopiridol derivative L868 276
is about ten times less active on cdk’s compared to
flavopiridol.
Binding of L868 276 to cdk2 is characterized by
predominantly hydrophobic and van der Waals interac-
tions with the same hydrophobic enzyme residues that
form the pocket for the adenine base in the ATP
complex structure [93]. However, there are more con-
tacts between the enzyme and the benzopyran ring of
deschloro flavopiridol (34 contacts) than were seen
between the enzyme and the adenine ring of ATP (26
contacts). Many of the contacts between L868 276 and
cdk2 are made by only three residues, Ile10, Leu83 and
Leu134, which form a total of 24 contacts, correspond-
ing to 43% of the observed contacts. Two of the cdk2
residues in contact with phenyl rings, His84 and Lys89,
are unique to the cdk2-L868 276 complex [92].
The shape complementarity of the contact surfaces of
both partners is described as solvent-accessible surfaces
that become buried upon ligand binding. In the cdk2-
ATP complex the buried areas of ATP (352 A, 2) and
cdk2 (435 A, 2) show a close fit. ATP is almost com-
pletely inaccessible to solvent and its buried surface
amounts to 80% of the buried surface in cdk2. Corre-
sponding values are 301 A, 2 and 399 A, 2 for L868 276
145
and cdk2, respectively, and the buried surface for
L868 276 amounts to 75% of the buried surface in cdk2
[92].
Flavopiridol has a chlorophenyl instead of the phenyl
in the L868 276 molecule. This modification increases
the kinase inhibition by a factor of about 10, probably
due to the new possible contacts that the chlorine
makes with residues Leu10, Phe82 and Leu93, increasing
the total number of contacts between flavopiridol and
enzymes to 61 [92].
When cytosolic soluble tumor cell extracts were
tested for proteins binding to immobilized flavopiridol
it was revealed [94] that cdk’s (i.e. cdk1, -2, -4, -7) bind
only weakly (possibly for steric reasons, because
flavopiridol via its hydroxy groups was bound to Sep-
harose 6B and one of the hydroxy groups of flavopiri-
dol mediates contacts with the enzyme [92]) when ATP
was absent but not in its presence. In contrast several
other proteins including cytosolic aldehyde dehydroge-
nase class-1 (ALDH-1) could be identified to bind
strongly to flavopiridol. ALDH-1 did not metabolize
flavopiridol and flavopiridol did not inhibit the enzy-
matic activity of ALDH-1. Nevertheless, the intracellu-
lar level of ALDH-1 correlated with the resistance of
tumor lung cells to flavopiridol [94].
Thus, there might exist still unknown intracellular
binding partners for flavopiridol. Noteworthy in this
context is the recent finding that flavopiridol is able to
decrease the stability of mRNA encoding VEGF in
mononuclear blood cells [95].
4. Characteristics of the antiproliferative activity of
flavopiridol
4.1. Induction of apoptosis
As has already been outlined (Section 3.1) expression
and function of cyclin dependent kinases and their
substrates are regulated and controlled in a very com-
plex way [10,20,76,96 –98]. In cancer cells this control is
obviously dysregulated either by loss of proapoptotic
proteins or by gain of functions of proteins inhibiting
apoptosis [99,100].
Flavopiridol seems to interfer with this dysregulated
system in various ways not only by directly inhibiting
cdk’s involved in cell cycle but also by inhibiting the
expression of cyclin D1, cyclin D3 and possibly as a
consequence of this activity by reducing expression of
cdk4 [91].
In consequence, by inhibition of cdk’s flavopiridol
may reduce or block function of antiapoptotic, cell
cycle promoting proteins, enabling their antagonists to
inhibit cell proliferation, to induce differentiation [101]
and/or to induce apoptosis. All these phenomena may
contribute to the considerable cytotoxicity, shown for
146 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170

flavopiridol [1,2]. Thus as shown in Fig. 1, by inhibition
of phosphorylation of the pocket proteins pRb, p107
and p130, flavopiridol is able to give rise to hypophos-
phorylated active pocket proteins able to bind to and to
inhibit cyclins (cyclin D, A, E), transcription factors
involved in promoting cell cycle (E2F, c-Jun, Myc) and
nuclear proteins (i.e. MDM-2) inhibiting oncogene sup-
pressor products (p53). Inhibition of all these proteins
has been shown to promote apoptosis [102 – 106]. In
addition, inhibition of cdk2/cyclin A by flavopiridol
inhibits phosphorylation and inactivation of E2F in the
late S-phase of the cell cycle that may lead to apoptosis
[107].
As expected flavopiridol is able to induce apoptosis
of tumor cells and normal cells in vitro (see Table 2) as
well as of tumor cells in vivo [108,7]. The percentage of
cells showing signs of apoptosis (chromatin condensa-
tion, internucleosomal cleavage, DNA fragmentation
and/or annexin V binding) after 6–48 h in vitro expo-
sure to 0.1 –0.4 mM flavopiridol varied considerably (see
Table 2). Most sensitive to flavopiridol’s activity to
induce apoptosis are human leukemia cells independent
of their origin (cultured cells, fresh patient’s isolates)
and type (B-cell type, T-cell type) followed by head and
neck tumors, breast carcinomas and non-small cell lung
carcinomas. Flavopiridol even induced apoptosis in
those tumor cells that were insensitive to apoptosis due
to DNA damaging agents (g-irradiation and bleomycin)
[108,7]. Induction of apoptosis by flavopiridol was ac-
companied by activation of caspase 3 and increase of
cleavage products of its substrate poly (ADP-ribose)
polymerase (PARP) (see Table 3).
In contrast to tumor cells, normal cells of different
types showed a considerable variation in their sensitiv-
ity to flavopiridol (see Table 2). Whereas human pe-
ripheral lymphocytes died after exposure to flavopiridol

Table 2
Induction of apoptosis in cell lines by Flavopiridol

Numbers Flavopiridol Number of cell lines with apoptosis (% of Ref.

(mM) apoptotic cells)

B10 10–30 \30–60 \60–90 \90

Tumor cells
T cell leukemia 3 0.1 3 Bible and Kaufmann [133];
Parker et al. [186]
B cell leukemia 1 0.3 1 Guedez et al. [137]
(mantel)
1 0.01–0.1 1 Parker et al. [186], Kitada et al.
[187]
3 0.4 3 König et al. [320]
(EBV+)
12 0.1–0.2 7 5 Byrd et al. [109], Kitada et al.
patients [187]
Head+neck 5 0.4 5 Patel et al. [108,7]
Breast 2 0.3 1 1 Schwartz et al. [119]
1 (low Erb–2) 0.3 1 Li et al. [216]
2 (high Erb–2) 0.3 2
NSC lung 7 0.1–0.5 7 Kaur et al. [9]; Shapiro et al.
[134,321–323]
Prostate 1 0.2 1 Parker et al. [186]
Gastric 2 0.3 1 1 Schwartz et al. [119]; Werner et
al. [324]
Oesophageal 4 0.1 1 3 Schrump et al. [135]
Bladder 2 1.0 1 1 Chien et al. [136]
Normal cells
Peripheral 9 0.1–0.2 9 Byrd et al. [109]
lymphocytes patients
Endothelial 1 0.1 1 Brüsselbach et al. [110]
(HUVEC)
Cortical neurons 2 1.0 2 Giovanni et al. [111]; Stefanis et
(rat) al. [127]
 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170
Table 3
Activation of caspase by Flavopiridol
Number Flavopiridol
Exposure time Concentration
(h) (mM)
Tumor cells
B-CLL 3 pat. 4–2 0.18
NSC-lung 1 4 0.1–1.0
7 \4 0.1–0.5
Breast 1 24 0.3
3 24 0.3
Gastric 1 24 0.3
Normal cells
Endothelial 1 12 0.5
(HUVEC)
a
PARP, poly(ADP-ribose) polymerase.
to a similar high degree as human leukemic cells [109],
endothelial cells were less sensitive than most of the
tumor cells [110]. Apoptosis of human endothelial cells
was accompanied by increase of caspase 3 and increase
of cleavage products of its substrate PARP [110]. Sur-
prisingly, the induction of apoptosis by flavopiridol in
endothelial cells was independent of expression of cy-
clin dependent kinases (cdk1, cdk2), which suggests a
mechanism of action beyond simple inhibition of cdk’s
[110].
In addition, embryonic cortical neurons are quite
resistant to treatment with flavopiridol alone [111].
No indications exist that the flavopiridol mediated
antiproliferative activity is more selective for trans-
formed cells per se than for normal cells. Thus lung
cancer cells, diploid lung fibroblasts and normal human
bronchial epithelial cells respond similarly to flavopiri-
dol [112].
However, cycling transformed cells including tumor
cells, especially those in the S-phase, undergo apoptosis
much earlier and to a higher degree than the corre-
sponding non-transformed cells. Recruitment of tumor
cells to S-phase can be done by treatment with cytostat-
ics (see Section 4.2). The mechanism of S-phase sensi-
tivity and selectivity of transformed cells may be related
to cyclin A-cdk2 inhibition by flavopiridol which, as
has already been pointed out in Section 3.1, results in
inappropriately persistant E2F activity. In transformed
cells, in which the E2F activity is already high, the
additional E2F activity may lead to cell death [112].
In conclusion flavopiridol is able to induce apoptosis
in tumor cells as well as in normal cells. Proliferating
transformed cells seem to be more sensitive to flavopiri-
dol than proliferating normal cells. Inhibition of cdk’s
by flavopiridol seems to be the prominent, but not the
only mechanism of action.
147
PARPa cleavage Ref.
products
Caspase-3
++ (]4 h) ++ (]24 h) Byrd et al. [109]
n.d. ++ Kaur et al. [80]
++++ Shapiro et al. [323]
++ ++ Motwani et al. [116,117]
++ ++ Li et al. [216]
++ ++ Motwani et al. [116,117]
n.d. ] 50% of total PARP Brüsselbach et al. [110]
4.2. Interaction with cytostatics
The percentage of apoptotic cells as well as the
degree of cytotoxicity could significantly be increased
by treatment of tumor cells with cytostatic drugs (taxol,
mitomycin, doxorubicin, etoposide, cisplatin, cytara-
bine or 5FU) 24 h prior to flavopiridol treatment
[113 –121] (see Table 4).
Simultaneous treatment showed a significant syner-
gistic effect only with antimetabolites (5FU, cytarabine)
[113,118 –121] and flavopiridol treatment 24 h prior to
treatment with cytostatics still showed synergistic ef-
fects with antimetabolites but abrogated considerably
the cytotoxic effect of taxol.
All the cytotoxic drugs used in these combination
experiments are known to induce apoptosis of tumor
cells in all phases of the cell cycle [122]. However, most
sensitive to apoptosis induced by cytotoxic drugs seem
to be cells in the G2/M-phase [123]. The mechanism of
the cell cycle independent apoptotic effect of cytostatics
seems to include damage of mitochondria, as measured
by the mitochondrial inner transmembrane potential
[122].
Flavopiridol’s mechanism of action in synergy with
cytostatic drugs seems to be different dependent on the
drug. Damage of mitochondria could be one of the
preconditions for promoting apoptosis by the subse-
quent treatment with flavopiridol. For instance in pacli-
taxel treated cells, flavopiridol is able to enhance
activation of caspases, thus intensifying paclitaxel in-
duced apoptosis [124]. Another precondition could be
the effect of cytostatic drugs on the expression of cdk’s.
Paclitaxel-induced apoptosis is preceded by a transient
mitotic block with elevation of cyclin B1 expression,
cdk1 kinase activity and MPM-2 reactivity, followed by
a decrease in these markers, hypophosphorylated Rb
148 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170

and induction of G1 markers but without cytokinesis
[124]. By inhibition of cdk1 kinase flavopiridol may
accelerate mitotic exit after paclitaxel therapy and
hereby enhance apoptosis.
In contrast, when administered together or before
paclitaxel, flavopiridol could override the paclitaxel ef-
fect of cdk1 kinase activation but may inhibit entry into
M-phase, a prerequisite for paclitaxel-induced apopto-
sis [124].
An other way of action of flavopiridol in synergy
with cytostatic drugs could be its direct interaction with
DNA, which may cause death of cells pretreated with
cytostatics [125].
The synergy in cytotoxicity of paclitaxel and
flavopiridol seems to offer an attractive clinical treat-
ment opportunity, i.e. a patient with prostate car-
cinoma, who failed prior paclitaxel treatment,
significantly responded to the combination of paclitaxel
and flavopiridol [11].
Synergy with flavopiridol has also been observed
with irinotecan. Exposure of human cancer cells to the
active metabolite of irinotecan induced p21 expression
but no apoptosis, whereas subsequent treatment with
flavopiridol reduced p21 expression, inhibition of cdk1
and cdk2 and apoptosis [126].
The effect of flavopiridol on enhancing mitomycin C
induced apoptosis seems to have quite a different mech-
anism. This synergic effect can at least partially be
reversed by pretreating the cells with the PKC activator
PMA under conditions proven to be associated with
activation of PKC. Thus, in combination with mito-
mycin C, flavopiridol mediates its synergistic effect at
least partly by inhibition of PKC [115 –117,120].
PKC has been proposed to be involved in phospho-
rylation and activation of the P-glycoprotein, one of the
drug transporter proteins, which can be overexpressed
in drug resistant cells (see Section 6).
However, pretreatment of tumor cells with flavopiri-
dol does not result in a synergism with most cytostatics

Table 4
Synergistic effect of Flavopiridol with cytostatics on tumor cells

Tumor cells Cytostatic (day 0) Flavopiridol treatmenta Ref.

+24 h 0 −24 h

NSC lung
Cytoarabine +++ 0 (+) Bible and Kaufmann [113]
A549 5-FU ++ + +++
Doxorubicin + (+) (+) 0
BCNU ++ 0 (+)
Etoposide ++ (+) (+)
Paclitaxel + − −
A549 Paclitaxel (−) n.d. 0 Edelman et al. [114]
Calu-1 Paclitaxel ++ n.d. ++
Gastric
MKN-74 Mitomycin +++ n.d. + Schwartz et al. [120]
Paclitaxel +++ − − Motwani et al. [117]
Paclitaxel +++ n.d. + Motwani and Schwartz [115]
Cisplatin +++ n.d. n.d. Motwani and Schwartz [116,117]; Werner et al. [324]
Gemcitabine +++ n.d. n.d. Jung et al. [325]
Pancreatic
Capan-2 Gemcitabine ++ n.d. 0 Jung et al. [236]
Colon
Hct 116 Irinotecan (active ++ n.d. n.d. Motwani and Schwartz [125]
metabol.)
Breast
MDA-MB-468 Mitomycin n.d. n.d. ++ Motwani and Schwartz [115–117]; Schwartz et al. [120]
MCF-7 Paclitaxel ++ (−) Motwani et al. [117]
MDA-MB-468 Paclitaxel ++ (−)
O6arian
Ov429, Ov432, SKOv-3, Cisplatin ++ Makhija et al. [160]
SKOv-3R
Osteosarcoma
Sa OS-2 Doxorubicin ++ Li et al. [138]
Sa Os-2/9 Doxorubicin 0

a
In relation to treatment with cytostatics.
 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170
(see Table 4). Thus it seems unlikely that inhibition of
PKC by flavopiridol could reduce expression and acti-
vation of P-glycoprotein.
An additional aspect of the synergism between cyto-
static drugs and flavopiridol is the prevention of en-
doreduplication and/or apoptosis.
Breast and colon carcinoma cells with intact G1-S
check points (p53 + / + , pRb + / + ) arrest in G1 with 4n
DNA content in response to spindle inhibitors like
paclitaxel, whereas cells with defective G1-S check-
points (p5 − / − , pRb − / − ) endoreduplicate, become
polyploid and display dysregulated and persistent acti-
vation of cdk1/cyclin B1 and cdk2/cyclin E kinase
activity. Surprisingly, flavopiridol prevents the dysregu-
lated and persistent activation of cdk kinases, en-
doreduplication and development of polyploids when
administered after paclitaxel. Thus flavopiridol could
have the potential to prevent the occurrence of neo-
plasia [127].
Neuronal cells undergo apoptosis when treated with
B-amyloid protein (ABP) or camptothecin [111,128].
Surprisingly, however, subsequent treatment with
flavopiridol did not enhance apoptosis (as shown for
tumor cells) but prevented loss of cytochrome C and
mitochondrial transmembrane potential as well as cas-
pase activation and processing [128] and provided a
long-term rescue from death [111,128]. In comparison,
general caspase inhibitors rescued neurons from this
rapid apoptotic death but had no effect on mitochon-
drial damage and delayed death. As cdk’s are known to
participate in death of neurons induced by DNA dam-
aging agents [129 –132] the protective effect of flavopiri-
dol for neuronal cells can be attributed to its potency to
inhibit cdk’s, i.e. cdk4 and cdk6, to inhibit hyperphos-
phorylation of pRb and p107 and to inhibit the tran-
scriptional activity of E2F/Dp [111].
In conclusion, flavopiridol shows a synergistic effect
with cytostatics on tumor cells, prevents development
of polyploid DNA content and protects neuronal cells
from apoptosis induced by cytotoxic compounds.
An explanation of this discrepancy could be that
dependent on whether the target cell is cycling or not,
the effect of flavopiridol might be different. Tumor cells
are programmed to proliferate and undergo apoptosis
after exposure to flavopiridol and inhibition of the
cdk’s expressed during cycling. In contrast, neuronal
cells are more programmed to be resting cells. DNA
damage in such resting cells stimulates expression of
cdk’s, which induce apoptosis if they are not inhibited,
i.e. by flavopiridol.
However, we also have to consider that the mecha-
nisms, underlying apoptosis and cytotoxicity induced
by flavopiridol in synergy with cytostatics, could be
much more complex.
149
4.3. Acti6ity in resting cells
The strong inhibiting activity on nearly all cdk/cyclin
complexes involved in cell cycle progression led to the
assumption that flavopiridol is mainly or even exclu-
sively active on cycling cells and that the cytotoxicity of
flavopiridol might be caused by inhibition of cdk/cyclin
complexes in cycling cells. Direct as well as indirect (via
cdk7/cyclin H) inhibition of cdk/cyclin complexes in
cycling cells should lead to reduced phosphorylation of
E2F as well as of the pocket proteins leading to in-
creased binding to and inhibition of E2F, other tran-
scription factors and nuclear proteins involved in cell
proliferation. It is assumed that such a blockage of
proteins involved in the cell cycle process could force
the cycling cells, especially tumor cells, into pro-
grammed cell death. If so, resting cells should be rela-
tively resistant to flavopiridol.
Several experiments (see Table 5) indeed demon-
strated that after exposure times of 24–28 h cycling
tumor cells are much more sensitive for flavopiridol
than the same density-arrested cells [9,86,87,110].
However, in contrast to the expectations according to
the proposed mechanism of action of flavopiridol, it
could also be shown both by using the same as well as
different tumor cell lines that resting tumor cells ex-
hibited the same extent of sensitivity during a 24 h
exposure to flavopiridol as proliferating cells
[109,110,133,134] (see Table 5).
Exposure of cells for a longer period of time (i.e. for
3 days or longer) increased the cytotoxicity by flavopiri-
dol significantly [1,5,86,87,110] and considerably re-
duced cell-type or laboratory-related differences in
cytotoxicity of flavopiridol between resting and prolif-
erating tumor cells [9,110,133].
Surprisingly, normal endothelial cells (HuVEC) were
sensitive to flavopiridol, irrespective whether they were
arrested by density or methionine deprivation or
whether they were proliferating or their expression of
cyclin A,-B, cdk1, cdk2, cdc25C, pRb or p27 was high
or low [110]. In contrast, embryonic cortical neurons
were resistant to flavopiridol [111].
These observations raise the question of whether the
mechanism that leads to cytotoxicity after flavopiridol
exposure is related to the cell cycle of the target cells
and to inhibition of cdk’s involved in cell cycle regula-
tion. The possibility that flavopiridol-induced cell death
involves other cellular targets must be considered [133].
In this respect it is interesting to note that flavopiridol
binds to genomic DNA to similar extent as ethidium
bromide and that the equilibrium dissociation constant
of the flavopiridol –DNA-complex is in the same range
observed for binding of the intercalators doxorubicin to
DNA [124]. However, results of these studies also
showed that flavopiridol does not inhibit topoisomerase
I and II.
150 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170

Table 5
Cell cycle dependence of the cytotoxicity of Flavopiridol

Flavopiridol Method for synchronizing IC50 (mM) Cell cycle Ref.

exposure time cells dependence
Resting cells Proliferating cells

Tumor cells
Breast 2 days Density arrest \0.3 – + Kaur et al. [80]
(MDA468)
6 days No 0.1 Carlson et al. [86]
24 h Aphidicolin (S-phase) 0.2
Nocodazole (M-phase) 0.2
Lung (A549) 24 h Density arrest :10.0 0.1 + Brüsselbach et al.
[110]
3 days Density arrest 0.6
24 days Starvation 0.3 0.3 − Shapiro et al.
[134,323]
24 h Density arrest 0.25 0.25 − Bible and
Kaufmann [133]
B-CLL (7 24 h No (Facs-analysis: DNA 0.18 0.18 − Byrd et al. [109]
patients) contents)
Normal cells
Endothelial cells 24 h Density arrest 0.1 0.09 − Brüsselbach et al.
(HuVEC) Methionine deprivation 0.08 [110]
Cortical neurons No \1.0 − Giovanni et al.
[111]

4.4. Acti6ity in relation to pRb expression
If cytotoxicity of flavopiridol on tumor cells is mainly
caused by inhibition of cdk’s, hypophosphorylation of
pocket proteins and inhibition of E2F and other tran-
scriptional activators involved in cell cycle, cells with
loss of function of pRb should be less sensitive to
flavopiridol.
However, no significant difference in the cytotoxic
activity of flavopiridol could be found between cell lines
expressing pRb and cell lines defective in Rb expression
(see Table 6) as was shown in esophageal cancer cells
[135], breast carcinoma cells [86,87] and in bladder
carcinoma cells [136]. Thus, the antiproliferative action
of flavopiridol does not seem to be dependent on the
presence of functional pRb. On the other hand, how-
ever, flavopiridol treatment induced a significant hy-
pophosphorylation of pRb [86 – 88] and a significant
reduction in the expression of cyclin D1 [88,135,137],
pRb and p107 [135]. Thus the phenotype of flavopiri-
dol-treated cells resembles that of cells in the G0/early
G1 phase of the cell cycle. Expression of p107 and
cyclin D1 were reduced by flavopiridol irrespective
whether the cancer cells were negative for pRb but
positive for p16 or vice versa [135].
Esophageal cancer cell lines that lack detectable Rb
protein expression showed a more pronounced rate of
apoptosis after flavopiridol treatment [135]. Similarly,
osteosarcoma cell lines lacking Rb protein were more
sensitive to the proapoptotic effect of taxol derivative in
combination with flavopiridol than osteosarcoma cells
transduced to express the Rb protein [138]. These ob-
servations are consistent with other data that have
demonstrated that Rb expression enhances resistance to
apoptosis mediated by diverse stimuli including IFNg,
radiation and cytostatics [139 –142].
Reduction of cyclin D1 expression after flavopiridol
treatment can at least partly be explained by the re-
duced level of hyperphosphorylated pRb. pRb is
mainly hyperphosphorylated by cdk4/cyclin D com-
plexes. Hyperphosphorylated pRb is proposed to in-
crease expression of cyclin D1 [143] and to produce a
feedback inhibition of cdk4/cyclin D activities by en-
hanced expression of the cdk4 inhibitor p16 [10,143].
On the other hand hypophosphorylated pRb binds to
and inactivates cyclin D and as a final consequence
leads to a reduced level of both proteins. In cells with
no pRb expression p16 is overexpressed and inhibits
complex formation between cdk4 and cyclin D
[144,145]. In addition flavopiridol has been shown spe-
cifically to repress the activity of the cyclin D1 pro-
moter [100]. Thus, in cell lines that lack cdk4
flavopiridol inhibited G1 progression to S phase by
inhibiting cdk6 activity as well as cyclin D1 expression
[100].
 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170 151

Overexpression of cyclin D is known to be associated However, cytotoxicity is correlated with suppression

with several types of cancer [46 – 48,146 – 148]. More-
over, transgenic mice overexpressing cyclin D1 spon-
tanously develop tumor diseases [149]. Reduction of
cyclin D1 level by antisense cyclin D1 [150] or wild-type
p16 gene [151] delivered via viral vectors mediate
growth inhibition, cell cycle arrest and apoptosis in
carcinoma cells. Thus it may be concluded that reduc-
tion of cyclin D1 expression may be one essential
mechanism by which flavopiridol induces its
cytotoxicity.
In tumor cells that lack cdk4/cyclin D1 complexes
and are negative for pRb and in which therefore the
regulatory interplay between cdk4, cyclin D, p16 and
pRb is lost flavopiridol seems to be active mainly by
inhibiting cdk2/cyclin E and cdk2/cyclin A complexes
without altering cdk2 levels or cyclin E and cyclin A
levels as dramatically as shown for cyclin D1 [86,87].
High expression of cyclin A has been shown to corre-
late with tumor agressiveness [50]. Inhibition of cdk2/
cyclin A reduces phosphorylation mediated inactivation
of E2F in the late S-phase of the cell cycle that may
lead to tumor cell apoptosis [107].
In summary cytotoxicity of flavopiridol on tumor
cells and normal cells is independent of the genetic
status of the target cells, i.e. whether they are express-
ing pRb or p16 or not.
of cyclin D1. Overexpression of cyclin D1 is known to
be associated with tumor diseases. In tumor cells that
are deficient in the expression cyclin D1, cdk4 and Rb,
flavopiridol may be active via inhibiton of cdk2 [86]
and inhibition of the phosphorylation and inactivation
of E2F.
4.5. Acti6ity in relation to p53 expression
Expression and function of p53 are in several ways
dependent on the activity of cdk’s. The question was,
whether flavopiridol is modulating the expression of
p53 and whether the cytoxicity of flavopiridol is depen-
dent on the expression of p53.
p53 plays a central role in the cellular response to
DNA damage, providing by induction of a cell cycle
delay or apoptosis a protective effect against tumorige-
nesis. Targets of p53 include genes associated with
growth control and cell cycle check points (e.g. p21
(WAF-1), GADD45, MDM2, EGFR, PCNA, cyclin
D1, cyclin G), DNA repair (e.g. GADD45, PCNA,
p21) and apoptosis (e.g. Bax, Bcl-XL, FAS, FASL)
[152].
Mutations of p53 are estimated to contribute to
around 50% of all cancers [153,154]. p53 is activated by

Table 6
Cytotoxicity of Flavopiridol in correlation with the genetic status for Rb and p16

Rb/p16 status No. Cytotoxicity Reduction of expression to No change Ref.

(IC50 mM) (100%)

B30% \30%, B80%

Tumor cells
Rb+/+ B-CLL 7 pat. 0.2 – – p27 Byrd et al. [109]
B-CLL (mantel) 3 0.3 Cyclin D1 – p27 Guedez et al.
[137]
Breast 1 0.3 Cyclin D1 Cyclin A, E cdk2, -4 Carlson et al.
[86]
1 0.3 Cyclin D1 Cyclin D3 Cyclin E, D2 Carlson et al.
[91]
Osteosarcoma 1 0.3 – – cdk2, p21 Li et al. [138]
NSC lung 4 0.2 – cdk2 cdk1 Kaur et al. [80]
Head+neck 1 0.1 Cyclin D1 Cyclin D3, E, Patel et al. [108]
cdk1, -2
Bladder (p16−/−) 2 B1.0 – – – Chien et al. [136]
Rb−/− Breast (cdk4−/−, 1 0.2 – Cyclin A, E cdk2 Carlson et al.
cyclin D1−/−) [86]
Oesophageal 2 0.1 Cyclin D1, p107 – – Schrump et al.
[135]
Bladder 2 B1.0 – – – Chien et al. [136]
p16m/m Head+neck 4 0.1 Cyclin D1 – – Patel et al. [108]
p16−/− Oesophageal 2 0.1 Cyclin D1, pRb, – – Schrump et al.
p107 [135]
Bladder 2 B1.0 – – – Chien et al. [136]
Normal cells
Rb++ Breast 1 0.2 Cyclin D1, D3 – n.t. Singh et al. [88]
152 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170
Fig. 2. Inhibition of cdk’s by flavopiridol may effect pocket proteins, p53 and bcl-2.
DNA damage (i.e. double strand breaks, mismatches
single stranded DNA) [155,156]. Its half-life time and
transactivation activity is increased by phosphorylation
through DNA-PK, MAP kinases, SAP kinases and
cdk’s [157]. Phosphorylation of the sites on the C-termi-
nal end of p53 by cdk’s has been shown to activate the
sequence specific binding of p53 in a manner specific
for the promoters of stress-responsive genes [158,159].
p53 induces the expression of and is inactivated by
MDM-2. Inhibitors of MDM-2 include hypophospho-
rylated pRb [76]. Thus, flavopiridol, by inhibiting the
phosphorylation of pRb, may promote inhibition of
MDM-2 and release of active p53. Active p53 in turn
can enhance expression of MDM-2 and of pRb [152].
On the other hand, by inhibition of cdk2 flavopiridol
may inhibit phosphorylation and activation of p53 (see
Fig. 2).
However, in spite of the many interactions between
cdk’s and p53 nearly all studies on different cell lines
and clinical isolates showed that the antiproliferative
activity of flavopiridol is clearly independent on the
genetic p53 status of the cell (see Table 7). Cells with
wild-type p53, inactivated p53 (by HPV-16-E6), mu-
tated p53 or without p53 expression responded equally
to flavopiridol (see Table 7). In p53 null ovarian car-
cinoma, transfection of p53 even enhanced the cyto-
toxic activity of flavopiridol [160] and, on the other
hand, deletion in p53 increased the resistance rate of
embryonic fibroblasts to flavopiridol [161].
In most cases flavopiridol had no effect on the level
of p53 expression. However, a significant reduction of
p53 expression could be seen in B cell lymphoma, which
might be the result of a higher degree of ubiquination
and degradation of the less phosphorylated p53, caused
by inhibition of cdk’s through flavopiridol.
Whereas the cytotoxic activity of flavopiridol in most
cases was independent of p53 expression, the synergistic
cytotoxic effect of taxol and flavopiridol (given 24 h
after taxol) was shown to be strongest in tumor cells
with deletion in p53 expression [114]. This indicates a
mechanism of synergistic action that is counteracted by
p53.
4.6. Acti6ity in relation to Bcl-2 expression
In view of the fact that targets of p53 include genes
of the bcl-2 family [152,162] the interesting question
was whether cytotoxicity of flavopiridol is correlated
with the expression of these proteins.
The Bcl-2 family of proteins consists of both in-
hibitors and promoters of apoptosis [163,164] via its
function as an ion channel and as an adapter or dock-
ing protein, e.g. for calcineurin and p53 binding protein
[165].
Inhibitors of apoptosis (including Bcl-2, Bcl-X, Bcl-
w, Bfl-1, Brag-1, Mcl-1 and A1) contain four domains:
1. The hydrophobic C-terminal transmembrane do-
main that allows their insertion into intracellular
membrane, e.g. mitochondrial membranes;
2. The pore formation domain formed by BH1 and
BH2
3. The ligand domain BH3 and
4. The docking domain BH4, by which Bcl-2 and other
inhibitors of programmed cell death (PCD) of the
Bcl-2 family can bind Raf-1 and other regulatory
proteins and target these proteins to mitochondria.
Promoters of apoptosis (including Bax, BAD, Bak,
Bcl-Xs, Bid, Bik and Hrk) lack at least one of these
domains, in most cases (except Bcl-Xs) the docking
domain BH4.
 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170 153

All pro-survival bcl-2 like genes are potentially onco-
genic, and some mutations probably increase their ex-
pression indirectly. In hematopoietic cells oncoproteins
such as Myb, Ras and AML1-ETO induce Bcl-2 ex-
pression [166,167] and that of Bcl-2 and A1 is often
elevated in myeloid leukemia and in esophageal, col-
orectal and stomach cancer, and reciprocal to p53 and
c-myc expression [168 – 171].
Pro-apoptotic Bcl-2 family members may act as tu-
mor suppressors. Loss-of-function mutation of Bax can
be found in human gastrointestinal cancer and some
leukemias [172 –175]. Activation of Bax expression by
p53 can provoke apoptosis [162,176,177].
All members of the Bcl-2 family form homo- or
selective pairs of heterodimers. Dimerization is per-
formed with the BH1, BH2 or BH3 domain and intra-
cellularly inhibited by phosphorylation of the different
proteins of the Bcl-2 family and/or by the action of
proteases [163,178].
Formation of heterodimers of apoptosis inhibitors
with promoters of apoptosis neutralize the antiapop-
totic effect of inhibitors. Thus, it depends on the ratio
of inhibitors and promoters in the cell, whether a cell
will survive and proliferate or whether apoptosis is
induced either via direct activation of proteases and/or
through the opening of the mitochondrial permeability
transition pores and the mitochondrial release of
protease activators [179 – 183].
Phosphorylation of pro-apoptotic members of the
Bcl-2 family seems to be performed by Raf-1. Raf-1 can
bind with its C-terminal catalytic domain to the BH-4
domain in Bcl-2. Via this binding, Raf-1 is targeted to
mitochondrial membranes, possibly allowing Raf-1
there to phosphorylate (and hereby to inactivate) BAD
and possibly other protein substrates promoting apop-
tosis [183].
For pro-survival members, phosphorylation may
both augment and suppress activity [183]. Bcl-2 may be
activated by Ser70 phosphorylation but inactivated or
otherwise altered by phosphorylation of several loop
sites, perhaps by Jun kinase (JNK) [184,185]. Sustained
activation of the JNK or p38 kinase pathways, perhaps
after caspase activation, has been implicated in
apoptosis.
Expression of Bcl-2 family members is induced tran-
scriptionally by certain cytokines and Bax is induced in
some cells as part of the p53 mediated damage response
[162].
Flavopiridol seems to downregulate Bcl-2 expression
in several B-cell leukemia cell lines [120,186,187] but
not in primary B-CLL cells from patients [109,186]. In
cell lines of ovarian carcinoma [120] and prostate car-
cinoma [186] the expression of Bcl-2 was likewise sig-
nificantly reduced (see Table 8).
As flavopiridol did not reduce expression of Bax, it
may be assumed that reduction of Bcl-2 by flavopiridol
in some cell lines could allow Bax and other proapop-
totic family member to induce apoptosis possibly via
activation of caspase-3 [80,109,110].
However, it is also evident from the results summa-
rized in Table 8 that reduction of Bcl-2 level is not an
essential requirement for the antiproliferative activity of
flavopiridol as there seems to exist no correlation be-
tween the cytotoxic action of flavopiridol and its po-
tency to reduce expression of Bcl-2.

Table 7
Cytotoxicity of Flavopiridol in correlation to the genetic status p53

p53 status No. IC50 (mM) Expression of p53 (%) Ref.

Tumor cells
p53+/+ B-CLL 5 pat. 0.2 B5 Byrd et al. [109]
1 0.1 B5 Parker et al. [186]
Breast (MCF-7) 1 B0.3 100 Carlson et al. [86]
Ovarian (p53 transduc.) 1 0.1 100 Makhija et al. [160]
NSCL lung 1 0.1 100 Kaur et al. [80]
Oeseophageal 4 0.3 100 Shapiro et al. [322,323]
p53m/m Breast 1 0.2 100 Schrump et al. [135]
B0.3 0 Carlson et al. [86]
NSCL lung 3 0.2–0.5 0 Shapiro et al. [322]
6 0.1–0.5 0 Shapiro et al. [323]
p53−/− Myeloid leukemia 2 0.1 0 Parker et al. [186]
T-ALL 1 0.1 0 Parker et al. [186]
prostate 1 0.2 0 Parker et al. [186]
ovarian 1 0.3 0 Makhija et al. [160]
Normal cells
p53+/+ Murine splenocytes 1 0.3 100 Byrd et al. [109]
p53−/− Murine splenocytes (null type) 1 0.3 0
154 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170

Table 8
Modulation of the expression of Bcl-2 family proteins by flavopiridol

No. Cytotoxicity IC50 Expression reduced to (%) Ref.

(mM)
Bcl-2 Bax

Tumor cells
Ovarian 1 0.1 0 n.d. Schwarz et al. [120]
Prostate 1 0.1 30 100 Parker et al. [186]
B-CLL 2 0.4 B50 100 Kitada et al. [187]; König et al. [326]
(unchanged)
B-CLL 1 0.4 :80 n.d. Schwartz et al. [120]
Breast (low Erb-2) 1 0.3 :80 ]100 Li et al. [216]
Breast (high Erb-2) 2 0.3
B-CLL 7 pat. 0.2 Â Byrd et al. [109]
Ã
1 0.2 Parker et al. [186]
Ã
Gastric 4 0.8 100 100 Schwartz et al. [120]
Ì
Oesophageal 4 0.1 (unchanged) (unchanged) Schrump et al. [135]
Ã
NSCL 4 0.2 Kaur et al. [80]
Ã
Mesotheliom 1 0.3 Å Waheed et al. [183]
Normal cells
Blood mononuclear 9 volunt. 0.2 100 n.d. Byrd et al. [109]
cells (unchanged)

On the other hand, neither transfection of tumor cells
to express Bcl-2 does provide resistance of those cells to
flavopiridol [161,188] nor does inhibition of expression
of Bcl-2 by antisense RNA increase cytotoxicity of
flavopiridol [188].
In summary, there exists no evidence that the expres-
sion of Bcl-2 is causatively involved in the mechanism
of flavopiridol induced cytotoxicity. Moreover, expres-
sion of Bcl-2 does not provide resistance of cells to
flavopiridol.
The fact that flavopiridol is not cytotoxic for cultured
embryonic cortical neurons but prevents camptothecin
induced loss of cytochrome C and mitochondrial
transmembrane potential as well as caspase activation
and processing [128,189] may support the assumption
that cytotoxicity caused by flavopiridol is not per se
induced by interference with Bcl-2, Bax and function of
mitochondria.
5. Inhibition of other kinases
The present data indicate that inhibition of cell cycle
cdk’s is most probably the main mechanism for the
proapoptotic and cytotoxic effect of flavopiridol on
cycling cells. However, its cytotoxic activity on resting
cells is difficult to explain on the basis of that
mechanism.
Possibly, inhibition of other kinases in addition to
cdk’s may be essential for this kind of antitumoral
effect. Although the inhibiting activity on EGFR ki-
nase, pp60 Src, PKC and Erk-1 is at least 50 times less
than on cdk’s, considering the intracellular concentra-
tion estimated to be achieved after systemic application
of flavopiridol, inhibition of such kinases may still
contribute to the proapoptotic and cytotoxic activity of
flavopiridol.
5.1. Interaction with receptors for the EGF family
The EGFR serves to regulate the proliferation of
multiple tissues during fetal development, non-repro-
ductive tissues in adult life, and multiple tissues during
pregnancy [190]. Overexpression of EGFR results in
EGF-dependent transformation [191,192]. Many tu-
mors of epithelial origin including breast, colorectal,
non-small cell lung, head and neck, ovarian, bladder
and prostate carcinomas show an aberrant, enhanced
and/or constitutive expression of EGFR [14,192] and
respond to EGFR binding growth factors (e.g. EGF,
TGFa, amphiregulin, heparin binding EGF, epiregulin
and betacellulin) [193]. Expression of high levels of
EGFR in tumors strongly correlates with a poor
prognosis.
The co-expression of EGFR binding growth factors
and EGFR frequently occurs in human carcinomas,
suggesting that autocrine activation of the receptor
might play a role in cancer cell growth [194 –196].
The c-ErbB-2 protein is a transmembrane protein
with substantial homology to EGFR. The normal func-
 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170
tion of the c-ErbB-2 protein is not completely known
[197]. The normal functions of the structurally related
c-ErbB-3 or c-ErbB-4 receptors are even less certain
[197 –200].
In tumors including stomach, prostate, breast, lung
(adeno carcinoma), pancreas and endometrium consti-
tutive activation by point mutations [201] or amplifica-
tion [193,202] and/or overexpression of the c-ErbB-2 or
the c-ErbB-3 proteins (prostate carcinoma) has been
found [14] and positively correlated with invasion and
metastasis [203,204].
In their inactive state the receptors for the EGF
family are monomeric. Receptor activation usually in-
volves dimerization induced by the binding of growth
factor polypeptides or an increase in the association
between already dimerized subunits.
Ligand binding brings into close contact tyrosine
kinase cytoplasmic domains resulting in the autophos-
phorylation and/or cross-phosphorylation [205] of sev-
eral receptor C-terminal tyrosine residues. Receptor
tyrosine phosphorylation generates sites for binding of
SH2 (Src homology) domain adapter and signal trans-
ducing proteins that function as substrates for the
phosphorylation by the receptor kinases and hereby
gain kinase activity [190,205 – 210].
The cytoplasmic domain of the EGFR owns six
phosphorylation sites. The T654 is located in the N-ter-
minal, juxtamembrane region. It is specifically phos-
phorylated by PKC [211] whereas the T669 is
phosphorylated by Erk1/2 [212]. Phosphorylation of
T654 inhibits signal transduction of the EGF receptor.
Thus the PKC plays the main cellular opponent of
ligand induced EGF-R-activation [213].
The cytoplasmic domain of the c-ErbB-2 also owns
one juxtamembrane site (T686) for phosphorylation by
PKC and three additional sites for autophosphoryla-
tion [214].
Flavopiridol has been shown to inhibit the kinase
activity of ligand-activated EGF receptors isolated
from cell membranes (A431 cells) [1,5]. Any interfer-
ence by flavopiridol with binding of the ligand to its
EGF receptor could clearly be excluded [5].
The direct inhibition of receptors for the EGF family
may be an additional mechanism by which flavopiridol
can inhibit proliferation of tumors. This effect could be
tumor selective insofar as many tumors show overex-
pression, amplification, constitutive or autocrine activa-
tion of such receptors.
In addition, through the inhibition of Erk-1 flavopiri-
dol may indirectly inhibit the activation of the EGFR
as well as the signal transduction pathway stimulated
by activated EGFR. In contrast, by the inhibition of
PKC flavopiridol may simultaneously inhibit phospho-
rylation of T654 and hereby block the inactivation of
the EGFR (see Fig. 3). It is quite possible that the
conflicting situation arising by blockage of inactivation
155
on the one site and direct and indirect inhibition of the
kinase activity of the activated EGFR on the other site
may force cells (especially tumor cells) into pro-
grammed cell death. In agreement with this assumption
it could be shown that flavopiridol can interrupt an
EGF-activated survival pathway [215].
An additional mechanism of action of flavopiridol
could be downregulation of c-ErbB-2 expression in
tumor cells. Breast carcinoma cells showed a 55–90%
decrease in c-ErbB-2 expression after exposure to
flavopiridol compared to untreated control [216].
Downregulation was much more pronounced in (wild-
type) parental cells compared to those stably trans-
duced to overexpress c-ErbB-2 [216]. However, after
flavopiridol treatment neither the dose dependent
growth inhibition nor the degree of apoptosis was
influenced by the status of c-ErbB-2 expression. Thus
ErbB-2 expression in breast cancer cells induces neither
resistance nor sensitivity to flavopiridol. However,
flavopiridol may reduce malignancy of breast car-
cinoma cells by reducing c-ErbB-2 expression.
5.2. Inhibition of receptor associated protein kinases
Cell membrane receptors lacking intrinsic kinase ac-
tivity need association with and activation of mem-
brane-associated kinases for signaling. Such receptor
associated protein kinases can be different depending
on the type of receptors [14]. However, there seems to
exist no complete restriction, i.e. several growth factor
receptors exhibiting intrinsic tyrosine kinase activity
have been additionally shown to form heterodimeric
complexes with multiple signaling and bridging
molecules via (tyrosine) phosphorylated Src homology
region interactions [197]. For example, the insulin re-
ceptor can be hyperactivated with the help of c-Src
[217]. Moreover, isolated c-Src SH2 domain can bind
activated EGFR specifically and directly [218,219] and
is required for EGF-dependent mitogenesis [220]. c-Src
can phosphorylate the Tyr-845 site of the EGFR in
addition to its autophosphorylation [219]. In addition,
the phosphorylation of the EGFR or of c-ErB-2 may
involve other Src family members, or the JAK kinases
[221].
On the other hand PTKs like c-Src can interact with
substrates independent from receptor [222 –224]. Bind-
ing of proteins of the Src family is mediated by their
myristilated N-termini (i.e. to the inner surface of the
plasma membrane), by their SH2 domains to sequences
containing phosphotyrosine and by their SH3 domains
to motifs rich in proline residues [225].
The Src family of PTKs includes pp60 c-Src, Yes
(expressed in a variety of tissues, especially epithelial
cells and brain), Fyn (mainly expressed in T- and
B-cells), Hck (mainly expressed in monocytes and gran-
ulocytes) Fgr (mainly expressed in monocytes) and Lck
156 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170
(mainly expressed in T-cells) [225]. As components of
the receptor system their stimulation initiates a cascade
of events leading to cellular proliferation [205,226].
Overexpression of proteins of the c-Src family is
associated with a considerable number of tumor dis-
eases [225] and may synergistically act with modified
growth factor receptor in its oncogenic activity
[219,225,227].
Inhibition of Src kinase activity is known to induce
arrest of mitotic progression of cancer cells [227].
Flavopiridol is able to inhibit Src and to a lesser degree
Lck but exhibits no relevant activity on Hck and Fyn
[3]. This implies that flavopiridol is not only directly
active on receptors for the EGF family but also on
receptors that lack intrinsic tyrosine kinase activity and
transfer their activation signal via the receptor associ-
ated protein kinase Src. Through this mechanism
flavopiridol could be able to inhibit proliferation of
tumor cells. In addition, although the IC50 of flavopiri-
dol for Lck is slightly beyond the threshold of 40 mM
(Table 1) the slight inhibition of Lck and the stronger
inhibition of pp60 Src by flavopiridol may explain its
strong apoptotic activity on normal lymphoid cells, on
human lymphoma and leukemia cells and its significant
Fig. 3. Inhibition of kinases involved in receptor activation and signal transduction.
immune suppressive effect observed in vitro and in vivo
on antigen as well as mitogen stimulated T-cells and
B-cells [4].
5.3. Inhibition of protein kinase C
Protein kinase C (PKC) is a family of serine/
threonine specific single chain protein kinases, consist-
ing of at least ten isoenzyme species. ATP binding sites
are highly conserved between isoenzymes and resemble
those of other protein kinases. The regulatory domains
differ between isoenzymes. In the ‘conventional’ isoen-
zyme family it contains binding sites for Ca2 + , for
anionic phospholipids, for diacylglycerol (DAG) and
also for phorbol esters [228].
DAG is the physiological activator of PKC and
physiological activation of PKC appears to involve its
translocation from the cytosol to the cell membrane
[229,230]. DAG is generated by various signaling path-
ways including those activated by binding of a variety
of cytokines, hormones, neurotransmitters and growth
factors to their respective receptors.
The signal transduction pathway that operates via
PKC appears to be essentially ubiquitous and regulates
 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170
many cellular processes, including cell proliferation
and differentiation [231,232]. The extraordinary diver-
sity of response to PKC activation can be explained
by the multiple gene products that constitute the
PKC family, their functional heterogeneity and their
tissue-selective distribution [231,233 – 236].
Significant increases of PKC expression has been
observed in human leukemias, melanomas and breast,
colon, prostate and lung cancers. The metastatic po-
tential, aggressiveness and/or growth rate of tumor
cells seem to correlate with their PKC activity [237 –
242].
Surprisingly in human colonic adenoma or car-
cinoma and in early emergence papillomas, PKC ac-
tivity is significantly reduced as compared to adjacent
mucosa tissue and mucosa in control subjects [243 –
246]. Nevertheless, downregulation of PKC in col-
orectal tumor cells [247] by antisense oligonucleotides
induced apoptosis or inhibited DNA synthesis indicat-
ing distinct roles for PKC in growth and survival
[236].
Many substrates of PKC have an essential function
in cell proliferation including receptors for the EGF
family, for transferrin, insulin and acetylcholin, the
signal transducing proteins, several transcription fac-
tors, DNA methyltransferase, ribosomal protein S6,
lamin B, telomerase and histone (type III) [248 –250].
PKC can substitute for or synergize with signal trans-
ducing proteins to promote transformation. Many of
the proteins that act at the signal transduction cas-
cade, such as Ras, Src and ErbB2 are known to in-
crease cellular DAG that activates PKC [251 –256].
Activated PKC can activate Src as well as Ras and
by that mechanism reinforce its own activation. Acti-
vated PKC is able:
1. To stimulate the Ras-Raf signal transducing path-
way that includes activation of Erk-1 [236,257 –259].
2. To activate the transcription factors AP-1 [260] and
c-jun [261,262] and those involved in activation of
the c-fos and c-jun genes [263]. Enhanced activation
of the fos promoter results into constitutive fos/jun
expression and renders the cells growth factor inde-
pendent [238].
3. To phosphorylate and inactivate IkB, the inhibitor
of the transcription factor NFkB [70,71,264,265]. In
many tumor types, overexpression or constitutive
activation of NFkB could be observed [14]. Expres-
sion of IkBa is known to induce apoptosis in tumor
cells overexpressing NFkB [266,267].
4. To phosphorylate both subunits (telomerase associ-
ated protein 1 (TEP1) and telomerase reverse tran-
scriptase (TERT)) and hereby to activate human
telomerase [248,268,269]. Telomerase is responsible
for the synthesis and maintenance of telomeric
157
DNA that together with proteins forms a protective
cap known as telomeres for eukaryotic linear chro-
mosomes [270 –272]. In most human cancers telom-
eres stop shortening because of the de novo
synthesis of telomeric DNA by activated telomerase
[273].Inhibition of PKC should lead to a reduced
activation of telomerase, a reduced synthesis and
maintenance of telomeric DNA and an inhibition of
tumor cell proliferation.
5. To phosphorylate DNA methyltransferase, which
alters gene expression by changing DNA methyla-
tion patterns [274].
The involvement of signal PKC in the transduction
pathway can regulate cell death mediated by a variety
of proapoptotic stimuli. The effects of PKC modula-
tors, however, vary significantly depending on the pro-
apoptotic stimuli [275 –277].
Nevertheless, the many ways by which PKC con-
tributes to signal transduction from the cell membrane
to the nucleus makes this enzyme an attractive target
for the search of inhibitors [276]. Flavopiridol inhibits
PKC to a lesser degree than cdk, but nevertheless at a
concentration that seems to be achievable in vivo
whithin the cells after application of non-toxic doses of
flavopiridol [2]. Via inhibition of PKC flavopiridol may
inhibit signaling of many receptors including receptors
for antigens, neuropeptides (bombesin, vasopressin),
endothelin, IL-8, adhesive glycoproteins (fibronectin,
vitronectin, laminin, collagen IV), peptide hormones
(GH, prolactin) and many cytokines and interleukins.
Such receptors transduce their activation signal via
receptor-associated kinase including pp60 Src and other
Src family members including FAK, Fps, Fes, Fer,
Jak1/2/3 or Tyk2. Such kinases activate PLCg, which
cleaves inositol phospholipids to generate inositol
triphosphate and diacylglycerol (DAG), which is the
physiological activator of PKC [228]. By inhibition of
PKC flavopiridol may block cellular signaling in a
central and decisive way and may block activation of
transcription factors (AP-1, c-jun) or expression of
transcription factors (c-fos, c-jun) or activation of in-
hibitors of transcription factors (IkB) involved in cell
cycle progression and apoptosis (see Fig. 3). Abroga-
tion of Bcl-2 expression may be one of the many
consequences of inhibition of PKC by flavopiridol
[275]. The inhibition through flavopiridol of PKC pos-
sibly in concert with the inhibition of cdk’s, EGF-R,
pp60 Src and/or Erk-1 may be the mechanism by which
programmed cell death could be induced.
If inhibition of PKC is important for induction of
apoptosis of tumor cells by flavopiridol, its way of
action should be different to the PKC inhibitor 7-hy-
droxy staurosporine, which prevents lamin B phospho-
rylation and degradation in and apoptosis of human
leukemia cells induced by camptothecin [230].
158 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170
6. Modulation of multidrug resistance
Resistance of tumor cells to chemotherapy limits its
therapeutic effect. Resistance can be mediated by
known as well as still unknown mechanisms. Resistance
phenotypes include the overexpression of drug trans-
porter proteins [278] like the P-glycoprotein (PG-170)
encoded by the multidrug resistance-1 gene (mdr-1 )
[279], the multidrug resistance protein (MRP-190) en-
coded by the mrp-1 gene [280 – 282] or the expression of
the breast cancer resistance protein (BCRP) [161]. Re-
sistance may also be caused by disruption of apoptosis
activation due to overexpression of cyclin D1, Bcl-2 or
inactivation of p53 [283 – 286].
Drug transporter proteins belong to the superfamily
of ATP binding cassette transporters [287 – 289], which
appear to cause cellular resistance to chemotherapeutics
such as anthracyclines, vinca alkaloids and taxol via
transport of those drugs across cellular membranes
[279].
Drugs with neutral charge can be transported by
PG-170 as well as MRP-190, whereas anionic com-
pounds are thought to be transported by MRP-190
[287,288,290] and cationic compounds by PG-170 [291].
Overexpression of MRP-190 induced by doxorubicin
revealed resistance of cells to anthracyclines, epipodo-
phyllotoxins and vinca alkaloids but not to antimetabo-
lites or platinum-containing drugs and they show only a
low level of resistance to taxol.
Overexpression of PG-170 can be induced by the
same types of drugs inducing overexpression of MRP-
190 but the spectrum of the resulting resistance to drugs
is slightly different. What determines whether MRP-190
or PG-170 will be overexpressed in a particular instance
is not yet clear [278].
Overexpression of drug transporter proteins and acti-
vation of proteins involved in signal transduction and
control of cell cycle and apoptosis are connected with
each other in many ways, i.e.
1. Expression of Ras and p53 proteins appear to stim-
ulate PG-170 expression [292], and cell lines resis-
tant to cisplatin have been shown to overexpress the
c-fos gene [293].
2. Moreover, c-Myc overexpression can reverse the
MDR phenotype [294] and the promoter of the
mdr-1 gene harbors a Raf-1 responsive element
[295]. Raf-1 is known to enhance cytoprotection by
Bcl-2 [165]. Raf-1 is activated by Ras and PKC is a
strong activator of Ras (see Fig. 2) [14].
Whether PKC is involved in MDR is controversial.
PKC seems to be directly involved in phosphorylation
and activation of the P-glycoprotein [296]. Phosphory-
lation by PKC of PG-170 seems to be correlated with
its drug transport activity [297]. Clonally selected MDR
cell lines exhibit overexpression of PKC [118,298,299].
Activation of PKC-a in cancer cells can induce multi-
drug resistance [300 –303] and transfection of cells with
the gene for PKC-a can enhance drug resistance several
fold [304,305].
In contrast, however, compounds known to reverse
MDR like verapamil, also promote phosphorylation of
P-glycoproteins [306]. Thus the possibility exists that
phosphorylation of P-glycoprotein can also inhibit
MDR.
Flavopiridol showed no cross-resistance to many cy-
tostatics (see Table 9), i.e. it was cytotoxic on tumor
cells irrespective of whether these cells were made resis-
tant in vitro to doxorubicin [1,161] or cisplatin [160] or
were clinical isolates from patients refractory to treat-
ment with chlorambucil, cyclophosphamide, vincristine,
doxorubicin, cisplatin and antimetabolites [109] or g-ir-
radiation and bleomycin [7,108].
Flavopiridol’s absence of cross-resistance in respect
to most of the conventional chemotherapeutic agents
may contribute to the clinically responses to flavopiri-
dol observed in patients with treatment refractory neo-
plasms and prior disease progression [11 –13].
One of the molecular mechanisms of this cytotoxic
effect of flavopiridol on drug resistant tumor cells could
be inhibition of PKC resulting in a reduced expression
and activation of drug transporter proteins.
However, the fact that pretreatment of tumor cells
with flavopiridol 24 h prior to the addition of cytostat-
ics does not in the case of most cytostatics result in
their enhanced activity argues against blockage of the
PG-170 protein by flavopiridol (see Section 4.2).
Moreover, colon carcinoma cell lines that have been
exposed to increasing concentrations of flavopiridol
and possess 8-fold resistance to flavopiridol showed an
increased drug uptake, no difference in the expression
of PG-170 but a 4-fold resistance to taxol [307].
On the other hand, a human ovarian carcinoma cell
line that spontaneously developed resistance to
flavopiridol (and to cisplatin) was just as sensitive as
the parental cells to cytostatics that are exported by
PG-170 or by MPR-190 [308].
Thus it is unlikely that flavopiridol is active on
resistant cells by direct action on the drug transporter
proteins PG-170 or MPR-190. Like other flavonoids
being cross-resistant to cytostatics flavopiridol is able to
increase the ATPase activity of MRP-190. But at low
concentration, flavopiridol is the only of these drugs
that inhibits the MRP-190 mediated transport of
daunorubicin and is cytotoxic for MRP-190 overex-
pressing cells. This cytotoxicity is slightly reduced (fac-
tor 3) compared to non-MRP-190 overexpressing cells
but still 103 × stronger than the cytotoxicity of the
other flavonoids [309,310].
Nevertheless, the intracellular concentration of
flavopiridol in the resistant cell line was lower com-
pared to that in the parental cell, a fact suggestive to a
drug transporter mechanism involved in cellular resis-
tance to flavopiridol [308].
 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170 159

Table 9
Activity of Flavopiridol on cells resistant to chemotherapy or radiotherapy

Tumor cells No. Drug resistance Degree/type IC50 mM (Flavopiridol) Ref.

Non-resistant Resistant cell line

cell line

Murine 1 Doxorubicin 110× 0.1 0.25 Sedlacek et al.

leukemia [1]
B-CLL 5 pat. Chlorambucil, prednisone, fludarabine, Clinically 0.12 (0.02–0.22) 0.15 (0.03–0.24) Byrd et al.
cyclophosphamide, vincristine, refractory [109]
doxorubicin, cisplatin
6 pat. fludarabine Clinically – 0.17 (0.09–0.21)
refractory
2 Doxorubicin MRP-1 (after 0.04 0.12 Hooijberg et al.
transfection) [309]
AML Doxorubicin MRP-1 0.09 0.13 Hooijberg et al.
[309]
Myeloma 1 Doxorubicin MDR 0.1–0.01 0.1–0.01 Schlegel et al.
[161]
Leukemia 1 Doxorubicin MDR 0.1–0.01 0.1–0.01 Schlegel et al.
[161]
Leukemia 1 Doxorubicin MPR-1 0.1–0.01 0.1–0.01 Schlegel et al.
[161]
Ovarian ca. 1 Cisplatin 6× – 0.3 Makhija et al.
[160]
Head and neck 4 g-Irradiation+bleomycin No apoptosis 0.01–0.07 B0.3 Patel et al.
ca. (IC95 =0.3) (IC95 =0.3) [108]
NSC lung ca. 1 7-Hydroxy-staurosporine No apoptosis – B0.3 Sugiyama et al.
[327]
Breast ca. 1 Mitomycin BCRP :0.1 :0.5 Schlegel et al.
[161]
Colon ca. 2 MRP transfection MRP 0.045–0.170 0.045–0.170 Smith et al.
[307]

Such mechanisms of resistance to flavopiridol, i.e.
overexpression of the drug transporter protein called
breast cancer resistance protein [161] or other mecha-
nisms, like overexpression of aldehyde dehydrogenase-1
or of glycogenphosphorylase [307], which are high
affinity cytosolic binding proteins for flavopiridol (but
do not enzymatically degrade it) [94] have already been
reported.
7. Antiangiogenic activity
In several test systems flavopiridol showed a signifi-
cant antiangiogenic activity (Table 10). Of special im-
portance may be the ability of flavopiridol to induce
apoptosis in resting as well as in proliferating human
endothelial cells [110]. The sensitivity of endothelial
cells to flavopiridol is higher (IC50:0.1 mM) than has
been reported for human normal fibroblasts (no induc-
tion of apoptosis) [311], for normal bone marrow cells
[6] and for normal peripheral lymphocytes [109].
Flavopiridol’s mechanism of action in inducing
apoptosis in resting endothelial cells is not completely
understood (as in other cells) and likewise it is not yet
known why endothelial cells are in vitro more sensitive
to flavopiridol than all the other normal resting and
proliferating cells tested.
Flavopiridol is also able to block completely in hu-
man monocytes hypoxia-induced VEGF mRNA and
protein expression by decreasing the VEGF mRNA
stability [95]. Neither transcriptional activation of the
6egf promoter nor the hypoxia inducible factor 1 activa-
tion sequence were affected and the constitutive levels
of VEGF mRNA were only slightly reduced by
flavopiridol. Via inhibition of PKC and fos promoter
activation flavopiridol might also reduce c-fos induced
VEGFD expression [312]. Another pathway could be
the inhibition by flavopiridol of Src, because activation
of Src is involved in the upregulation of VEGF expres-
sion [313].
The fact that flavopiridol is cytotoxic to endothelial
cells as well as inhibits hypoxia-induced VEGF expres-
sion suggests that inhibition of tumor angiogenesis
could play a considerable role in the antitumoral activ-
ity of flavopiridol [110].
Indeed, in the in vivo mouse matrigel model of
angiogenesis flavopiridol was more potent than the
known anti angiogenic compound TNP-470 in inhibit-
160 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170

ing neovascularization. Flavopiridol also showed at
least equivalent activity to TNP-470 in reducing mean
tumor volume in nude mice bearing human colon car-
cinoma xenografts [314].
The antiangiogenic effect of flavopiridol is supported
by its potency to decrease MMP activity of tumor cells
(i.e. breast carcinoma expressing low c-ErbB-2 or trans-
duced to express high c-ErbB-2) and to inhibit their
invasion through matrigel [216]. By secretion of degra-
dative enzymes tumors support invasion and attach-
ment of endothelial cells [315].
Surprisingly, no side effects of flavopiridol on the
vasculature have been reported from preclinical as well
as clinical studies [2,12,13], although flavopiridol is
similarly active on both cycling and non-cycling en-
dothelial cells. It may be possible that in vivo normal
blood vessels are less accessible for flavopiridol due to
thicker layer of bloodborne macromolecules that may
inhibit transmembrane transport of flavopiridol into
endothelial cells. In contrast, several clinical studies
have observed a high incidence of venous thrombotic
events that can be caused by a direct action of flavopiri-
dol on endothelial cells.
Proangiogenic factors in tumors are known to induce
downregulation of adhesion molecules such as ICAM-
1, ICAM-2, VCAM-1, E-selectin and CD34 on en-
dothelial cells in the tumor vasculature and to induce
resistance to inflammatory signals such as TNFa, IL-1
and IFNg [316 –319]. In addition, antiangiogenic fac-
tors like platelet factor 4 (PF4), thrombospondin-1 and
g-interferon inducible protein-10 can prevent or reverse
downregulation of the expression of adhesion molecules
on endothelial cells [317]. It may well be that a reduced
expression of adhesion molecules in the tumor vascula-
ture may enhance the sensitivity of tumor endothelial
cells to flavopiridol.
The fact that in patients hypotension is the dose
limiting toxicity of i.v. infused flavopiridol in combina-
tion with antidiarrheal prophylaxis [12,13] may indicate
that flavopiridol could damage the endothelial cell lin-
ing of the cardiovasculature at higher dosages.
Altogether, in addition to direct its direct cytotoxicity
for tumor cells flavopiridol may also inhibit tumor
growth by inhibition of tumor angiogenesis as well as
detoriation of tumor endothelial cells and tumor vascu-
lature. As antiangiogenic compounds are known to
enhance in vivo the antitumoral activity of cytotoxic
drugs, the combination of flavopiridol with cytostatic
drugs might provide therapeutic advantages in tumor
diseases.

Table 10
Antiangiogenic activity of Flavopiridola

Test systems Results Comments Ref.

In 6itro
Cytotoxicity for
endothelial cells
Resting cells IC50: 0.08–0.1 Not dependent on expression of cdk1, Brüsselbach et al. [110]
cdk2, cyclin A, -B, p27, pRb
Proliferating cells IC50: 0.09
Downregulation of
VEGF expression
Normoxic M ¥ Slight effect Reduction of mRNA half-life Melillo et al. [95]
Hypoxic M ¥ Complete blockage
Reduction of MMP
activity
Breast ca. Â Decreased levels of MMP Unclear, whether decline of MMP is a Li et al. [216]
(low Erb-2) Ã after 0.3 mM flavopiridol direct effect or caused by
Breast ca. Ì downregulation of Erb-2
(high Erb-2) ÃÅ
In 6i6o
Matrigel model Reduction of angiogenesis  Robinson et al. [314]
(mouse) Ì Equivalent to TNP-470
Colon carcinoma Tumor growth inhibition Å Robinson et al. [314]
xeno(mouse)
Clinical studies
Phase I study Hypotension catheter related Senderowicz et al. [12,13];
thrombosis induction of inflammatory Werner et al. [14]
markers

a
Senderowicz et al. [12]; Werner et al. [13].
 H.H. Sedlacek / Critical Re6iews in Oncology/Hematology 38 (2001) 139–170
8. Conclusion
Flavopiridol is a strong inhibitor of cyclin dependent
kinases (cdk-1, -2, -4, -6, -7) and less active on receptor
tyrosine kinases (EGFR), receptor associates tyrosine
kinases (pp60 Src) and on signal transducing kinases
(PKC and Erk-1).
Although the inhibiting activity of flavopiridol is
strongest for cdk, the cytotoxic activity of flavopiridol
is not limited to cycling cells. Resting cells are killed,
too. This indicates that inhibition of cdks involved in
the control of cell cycle is not the only mechanism of
action. Inhibition of cdk’s with additional functions
(i.e. involved in the transcriptional control of the ex-
pression of cyclins or transcription and/or function of
proteins that do not control the cell cycle) could con-
tribute to the antitumoral activity of flavopiridol. In
addition, direct and indirect inhibition of receptor acti-
vation (EGFR) and/or a direct inhibition of kinases
(pp60 Src, PKC, Erk-1) involved in the signal transduc-
tion pathway could participate in the antiproliferative
activity of flavopiridol.
From pharmacokinetic data in patients it can be
concluded that the inhibitory activity (IC50) of
flavopiridol on these kinase is in the range of concen-
trations that might be achieved intracellularly after
systemic application of non-toxic doses of flavopiridol.
However, no in situ data from flavopiridol treated cells
have been published yet that prove that by inhibition of
EGFR, pp60 SRC, PKC and/or Erk-1 (in addition to
inhibition of cdk’s) flavopiridol is able to induce apop-
tosis. Thus many questions regarding the detailed
mechanism of antitumoral action of flavopiridol are
still open. Nevertheless, the assumption seems to be
attractive that the strong antitumoral activity of
flavopiridol on cycling and resting malignant as well as
on normal cells could be caused by a complex mecha-
nism. Prominent seems to be the inhibition of cdk’s
involved in control of the cell cycle and/or activation of
transcription. Inhibition of receptors and kinases in-
volved in signal transduction may support the antitu-
moral effect of flavopiridol. This antitumoral activity
shows
1. High rate of apoptosis, especially in leukemic cells
2. Synergy with the antitumoral activity of many
cytostatics
3. Independence of its efficacy on pRb, p53 and Bcl-2
expression
4. Lack of interference with the most frequent multi-
drug resistance proteins (P-glycoprotein and MRP-
190) and
5. A strong antiangiogenic activity.
Based on these pharmacological data it can be con-
cluded that flavopiridol could be therapeutically active
in tumor patients
161
1. Independent on the genetic status of their tumors or
leukemias (i.e. mutations of the pRb and/or p53,
amplification of bcl-2)
2. In spite of drug resistance of their tumors induced
by first line treatment (and caused by enhanced
expression of multidrug resistance proteins)
3. In combination with conventional chemotherapeu-
tics given prior to flavopiridol and
4. Due to a complex mechanism involving cytotoxicity
on cycling and on resting tumor cells, apoptosis and
antiangiogenic activity.
In consequence, flavopiridol is a highly attractive,
new antitumoral compound and deserves further eluci-
dation of its clinical potency.
Reviewers
Professor Dr H.J. Broxterman, Academisch of Medi-
cal Oncology, PO Box 7057, NL-1007 MB Amsterdam,
The Netherlands.
Walter M. Staler, M.D., Assistant Professor of
Medicine, Section of Hematology/Oncology, University
of Chicago Medical center 5841 S. Maryland Avenue,
MC 2215, Chicago, IL.
Professor Cr G. Eisenbrand, Universität Kaiser-
slautern, Fachbereich Chemie, Gebäude 52/322 Erwin-
Schrödinger-Strasse, D-67663 Kaiserslautern, FRG
Germany.
Acknowledgements
The author is indebted to Manuela Rogala for her
skillful secretarial assistance in preparing the
manuscript.
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Biography
H.H. Sedlacek — Education: Veterinary Medicine
(University of Giessen); DVM, Endocrine Pharmacol-
ogy (1968). Awards: Adjunct Professor for Tumor Biol-
ogy, Medical School, University of Marburg (1994).
Occupational activities: Schering AG; Berlin/Bergka-
men 1969 –1971 Immunotoxicology; Behringwerke,
Marburg; 1972 –1983, Head of Immunopharmacology
and Tumor Pharmacology; 1984 –1991 Director of On-
cology Research; 1991 –1996, Head of Strategic Plan-
ning of R&D; Hoechst Marion Roussel/Aventis
Pharma Germany, 1997 –2000, Coordinator of Gene
Therapy R&D.
Major achievement: Essential Contribution to Dis-
covery and Development of Flavopiridol (Innovation
Award of German Industry in 1998).