Associate Professor

Basic Dental science Department

Faculty of Dentistry, Princess Nourah Bint
Abdulrahman University
 Introduction

Oncogene

Tumor suppressor genes

Causes of DNA damage

Aim of the work

CONTENT Materials and methods

Result

Conclusion

Recommendation

Conclusion
Introduction
▪ Cancer is the uncontrolled growth and spread
of cells which affect almost any part of the
body.
▪ The growths often invade surrounding tissue
and can metastasize to distant sites.
▪ Cancer is a consequence of mutations in
genes that control cell proliferation,
differentiation and cellular homeostasis.

▪ These genes are classified into two

categories : oncogenes and tumor
suppressor genes.
ONCOGENE
Any gene that encodes a protein able to
transform cells and induce cancer in human or
animal.

TUMOR SUPPRESSOR GENE

A protein that slows or inhibits cell
proliferation and prevents damaged or
abnormal cells from becoming malignant.
 BALANCE between oncogene and TSG
is mandatory for healthy living cells

Overexpression of oncogenes and loss of tumor suppressors

are the dominant cause of cancer formation.
ONCOGENES
Proto-oncogenes are normal cellular genes that are expressed
during normal growth and developmental processes.

Many oncogene are derived from normal cellular genes

(i.e., proto-oncogenes).

Conversion or activation of proto-oncogenes

into an oncogene generally involves a gain-
of-function mutation.
Mechanisms of production of
oncogenes from proto-
oncogenes are:
Point mutations in a proto-
oncogenes

Localized reduplication (gene

amplification)
 PROTO-
ONCOGENE
ONCOGENE

CANCER
Ras (HRas, KRas, and NRas) is responsible for
turn on many genes involved in cell growth,
differentiation and survival.

Growth factors (EGFs, FGFs,….)

overexpression (known as upregulation or
amplification) have been associated with a
number of cancers

Cyclin-dependent Kinases : critical regulators

of cell cycle progression.
 TUMOUR
SUPPRESSOR
GENES
Tumor suppressor genes are normal genes
that control cell cycle, cell division and
apoptosis (programmed cell death)
Rb
P53
P21
Rb

Retinoblastoma gene
“ first tumor
suppressor gene”
The first identified TSG.
RB is absent/mutated in at least one-third
of all human tumours.
It is associated with an inherited eye tumor.
It is an important regulator for cell cycle
progression (G1 exit block).
Rb is located on long arm (q) of
chromosome 13.

13q14
The most common
intraocular malignancy
in children.
leukocoria due to the
reflection of light by the
white mass in the
fundus.
p53

“Guardian of the
genome”
Wild type
or normal
p53 P53 is a key
protein TSG
protecting us
from
Mutations cancer
in p53 are
responsible for
50%
of all the human
cancers
The name p53 was given in 1979 describing
its molecular mass which is 53-kilodalton (kDa)
nuclear phosphoprotein and consists of 393 amino
acids.

(wild type or normal p53 protein).

It is located on the short arm (p) of chromosome 17.

17p13.1
p53 plays a major role in
repair of damaged DNA
It is essential for regulating
cell division and preventing
tumor formation, it has
been nicknamed as the
“guardian of the genome”
P53

Modulation of the DNA

damage response
Cell cycle arrest /
senescence

Apoptosis

Control of genome
integrity

DNA Repair
P53 mutations resulting in formation of
impaired proteins unable to bind DNA
effectively.
The damaged cells continue to grow and
divide in an unregulated way, which can
lead to cancerous tumors.
 Genotoxic and cytotoxic effect of Cone
Beam Computed Tomography (CBCT)
on exfoliated buccal epithelial cells

This study was accomplished to evaluate

genotoxicity and cytotoxicity in exfoliated buccal
mucosa cells of adults following CBCT exposure.
Materials &Methods
Patient selection:
The study was conducted on 30 healthy
males. The patients were recruited from
outpatient’s clinic of Faculty of Dentistry
Pharos University in Alexandria from May
2017 till December 2017 whom referred to
CBCT for diagnostic purposes. The patients’
age ranged between 25 to 40 years.
Exclusion criteria:
Systemic disorders.
Patient taking medications.
Alcohol or tobacco consumers.
Patients subjected to CBCT one month
prior to the study.
Careful intraoral examination was performed
including inspection and palpation of lips
buccal mucosa, vestibular mucosa, hard and
soft palate, tongue and floor of the mouth.
Patient with red and/or white colored or
pigmented lesions were excluded from our
study.
Informed consents were obtained from all the
selected patients.
Scanning protocol:
Patients were subjected to CBCT using J Morita CBCT
unit ( J. Morita, Corporation, Kyoto, Japan), referred to
CBCT for diagnostic purposes.
Imaging Software Included i-Dixel 2.0 software
operated at 84 Kilovoltage (kVp) and 9–14
Milliamperage (mA) with a voxel size of 0.16 mm,
exposure time of 6 seconds, and field of view (FOV) of
80x100 mm for all patients.
Cells collection and slide preparation:
Exfoliated buccal cells were collected
immediately before CBCT and after 10 days.
Each patient was asked to rinse with tap water
in order to remove any debris from the oral
cavity.
Exfoliated oral epithelial cells were collected
by scraping the cheek mucosa with a moist
wooden tongue depressor.
Cells collection and slide preparation
cont.:
The obtained cells were smeared on a
sterile glass slide.
Then, the smears were fixed with a
95% ethanol and stained with
Papanicolaou stain.
Cells collection and slide preparation:
Day 10 was chosen on the basis of the rapid
turnover of the epithelial cells that take 7–16 days to
emerge to the surface and to exfoliate.
The buccal mucosa is the preferred target that
accurately reflect the cytotoxic changes and genomic
instabilities in epithelial tissues due to its high
turnover rate that brings the cells to the surface.
Cytological analysis:
The smears were examined blindly by the
authors in randomly selected microscopic
fields at a magnification of x 400 to detect
the micronucleus and other cytological
alterations.
At least 1000 exfoliated epithelial cells in
each smear were counted.
The micronuclei were counted following Tolbert
et al. parameters for identifying micronucleus.
Other cytological changes were examined in
terms of nuclear alterations and different
chromatin status as pyknosis, condensed
chromatin, karyorrhexis and karyolysis.
The The micronucleus is a small extranuclear body
formed during cell division, either due to
chromosomal aberrations or improper function of
the mitotic spindles.
Therefore, micronucleus assay is a well-known
technique in monitoring recent exposure of
individuals to genotoxic agents, such as chemicals
and ionizing radiation.
Statistical analysis:
The Kolmogorov- Smirnov, Shapiro and
D’agstino tests were used to verify the
normality of distribution of variables.
Normally quantitative data were expressed
in mean ± standard deviation (SD).
Results
 The formation of micronucleus is In addition to the micronucleus, cytotoxic
A photomicrograph
attributed to chromosomal damage of exfoliatednuclear
buccalalterations included
epithelial cellspyknosis,
after
that could condensed chromatin and karyorrhexis which
CBCTnotexposure
be included in the nuclear
shows changes. indicated
a and b:cellular
micronuclei
daughter nuclei by the end of the
(black arrows), c: pyknoticdeath
mitotic process.
nuclei (blue arrows),
by apoptosis d:necrosis.
rather than
karyorrhexis (green arrow). (a-d: Pap stain, x400)
The genotoxic biomonitoring of this
study revealed that the frequency of
micronuclei in the exfoliated buccal
cells were increased significantly
after exposure to CBCT which
indicated a chromosomal damage .
The significant increase of the micronuclei as a
genetic damage biomarker after CBCT exposure
highlights the consequences of accumulation of
these genetic damages within the epithelial cells.

It is well known that such alterations are considered

as the fundamental cause of development of
premalignant and malignant lesions.
Recommendation
Further cytological Smear examination is
needed on prolonged interval more than 10
days to determine the time of complete
repair.
 Impact of
Radiation on DNA
damage
Reactive oxygen species (ROS)

Replication errors

X ray

UV light

Alkylating agents
 RADIATION
Indirect action
Direct action

Free radicals
DNA damage
DNA damage

Double strand Single strand

breaks breaks
Ionizing radiation can cause single-strand
breaks SSB in DNA (predominate) or
double–strand break DSB
(a relatively small percentage) .
Within the highlights of the results of the
present study, the authors concluded that
CBCT has inevitable health side effects
that should be taken into consideration,
and it must be used following the ALARA
(As Low As Reasonably Achievable)
principle, owing to the ability of CBCT to
induce cellular death.
 Ionizing radiation is a threat to human cells and
ultimately to the entire organism

This threat begins with the induction of DNA damage,

which may then be converted into permanent genetic
change. Ionizing radiation is an activator of onocogenes
and an inactivator of tumor-suppressor genes