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SDLS 2008

File Created on 10/14/2005 11:08 AM Transcriber(s): Kari, Joy, Mae Editors: JC Tayco, Mark Lomboy, Pre Ausan, Erika Mendoza XENOBIOTICS, CANCER, ONCOGENES, AND GROWTH FACTORS Xenobiotics  compounds that are considered foreign to life (nonnutrient compounds)  usually in pro-mutagen/pro-carcinogen form  are hydrophobic in nature, it tends to accumulate in the cell membrane.  examples: - therapeutic drugs ex: Rifampin, Cyclosporine - pesticides ex: DDT - pollutants ex: benzo-a-pyrene  Strategy: convert the hydrophobic compound to hydrophilic compound A closer look xenobiotics… on the given examples of Topic: Xenobiotics Lecturer: Professor Patricia Generoso No. of pages: 5 -

Detoxicol

Medicine for the intoxicated

catalyzed by members of enzymes monooxygenase (family of oxidoreductase) or cytochrome P450. Substrates are generally liphophilic and are rendered to become hydrophilic by hydroxylation May involve deamination, dehalogenation, desulfuration, epoxidation, peroxygenation and reduction.

RH + O2 + NADPH + H ---> ROH + H Phase II the hydroxylated or other compounds produced in phase I are converted by specific enzymes to produce to various metabolites involves the process of conjugation with glucoronic acid, sulfate, acetate or glutathione, or certain amino acids, or by methylation.

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Benzo-a-Pyrene - 1st carcinogen found - comes from food that is burned/charred (eg.tutong,b-b-q) - binds to guanine (at N7), leading to apuniric sites. Cadmium - found in battery and also cigarette smoking - binds to phosphate group -> DNA has a hard time unwinding -> interruption in transcription and translation Lead - leads to change in comformation of DNA Note that: - DNA structure is stable but is vulnerable during replication and transcription - Guanine—is the most reactive base - 80% of cancers are caused by chemical carcinogens  2 Phases of xenobitic metabolism: - Phase I - Metabolism by cytochrome p450  hydroxylation  RH + O2 + NADPH + H+ ---> ROH + H20 + NADP - Phase II - Conjugation - addition of another group to make the compound more hydrophilic From Harper’s 25th edition: Metabolism of Xenobiotics can be divided into two phases: Phase I major reaction involved is hydroxylation

Purpose of the two phases of metabolism: 1. increase their water solubility (polarity) 2. facilitate excretion Cytochrome p450 is a family of enzymes  At least 150 types of isoforms  RH + NADPH (electron donor) + O2 ------> ROH + NAPD + O2  Difference among isoforms is the use of R - Phenacetine - precursor of acetaminophen - Benzo-a-pyrene - a pollutant - Methamphetamine - a stimulant - Cardizine - drug for heart problems Nomenclature and Characteristics  CYIIA1 (CYP - II - A - 1 ) Family subfamily 1st member

 Monooxygenases - O2 is split, so one molecule of the oxygen goes into
the substrate, and the other into H2O Heme proteins (same type of heme as in hemoglobin and myoglobin but difference between the two hemes is that heme in hemoglobin and myoglobin only carries oxygen). - Heme in Cytochrome P450 can carry as well as split oxygen Most p450 metabolize first in the liver Found in smooth endoplasmic reticulum Uses NADPH Peak absorption of p450 is at 450 nm Drug Interaction - CYP2C9 (Warferin) -----------> Phenobarbital - CYP2E1 -------------------------> Ethanol - CYP1A1 ------------------------> PAH

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    

PHASE II - Conjugation Reactions

 Glucuronidation - UDP - glucoronic acid - 2 acetylamino fluoride, aniline, benzoic acid, phenol,
steroids

radiation Polycyclic hydrocarbons in soot, tar, oil 2Naphthylamine 1-naphthylamine Benzidine; 4aminobiphenyl Asbestos

etc. chimney sweepers, manufacturers of coal gas, many other groups of exposed industrial workers chemical workers, rubberbladder Workers, manufacturers of coal gas chemical workers asbestos workers shipyard and insulation workers sheep dip manufacturers, gold miners, some vineyard workers and ore smelters makers of ionexchange resins Workers with glues, varnishes, etc. poison gas makers

scrotum, skin bronchus

 Sulfation - PAPS -

(adenosine 3’ phosphosulfate) Alcohol, arylamine, phenols

phosphate

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5’

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 Glutathione - electrophilic xenobiotics  Acetylation - x + acetyl CoA -----> Acetyl - x + CoA - Isoniazid  Methylation - S-Adenosylmethionine
Cytochrome P450 GSH S-transferase or epoxide hydrolase

bladder bronchus, pleura, and peritonium skin and bronchus

Arsenic

Xenobi otics

Reactive metabolite

Nontoxic metabolite

Covalent binding to macromolecules

Bis (chloromethyl) ether Benzene Mustard gas

Bronchus marrow (leukemia) bronchus, larynx, nasal sinuses liver (angiosarcoma) bladder bronchus, pleura, and peritonium skin and bronchus

Cell injury

Hapten Antibody production Cell injury

Mutation

Cancer

Vinyl chloride Benzidine; 4aminobiphenyl Asbestos

PVC manufacturers chemical workers asbestos workers shipyard and insulation workers sheep dip manufacturers, gold miners, some vineyard workers and ore smelters makers of ionexchange resins workers with glues, varnishes, etc. poison gas makers

Protooncogene  gene whose protein product has the capacity to induce cellular transformation given it sustains some genetic insult Oncogene  gene that has sustained some genetic damage and, therefore, produces a protein capable of cellular transformation  Gene with potential to cause cancer Difference between Normal and Cancer Cells 1) Cancer cells are less subject to feedback mechanisms that control normal cell division, both in tissues and in culture. 2) Cancer cells, as a population, can go on dividing indefinitely * Increased levels of a Normal Cellular Protein Can Destroy Normal Cell Growth Regulation Chemical and Physical Carcinogenesis Table: Occupational Cancers Agent Ionizing radiations radon x - rays, radium radium Ultravolet Occupation certain underground miners (uranium, fluorspar,etc.) radiologist, radiographers luminous dial painters Farmers, sailors, Cancer site bronchus skin Bone Skin

Arsenic

Bis (chloromethyl) ether Benzene Mustard gas

Bronchus marrow (leukemia) bronchus, larynx, nasal sinuses liver (angiosarcoma)

Vinyl chloride

PVC manufacturers

Classification of Chemical Carcinogens Chemical carcinogens are of synthetic (“man made”) or natural origin, are extremely diverse in structure without any common feature, and are classified into two Categories:  direct-acting (DNA-reactive, activation independent, genotoxic) carcinogens that bind covelently to cellular genomic DNA and are mutagens;  procarcinogens (activation dependent) that require metabolic conversion to metabolites (“ultimate

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carcinogens”) capable of transforming cells and inducing tumors. Procarcinogens are among the most potent chemical carcinogens. Major Chemical Carcinogens A. Pro-carcinogens (require metabolic activation to “ultimate carcinogens”)  benzanthracene (first pure carcinogen)  3, 4- benzpyrene (isolated from coal tar)  3 - methylcholanthrene (prepared from a steroid, deoxyxholic acid)  7, 12 - dimethylbenzathracene (most potent carcinogen) B. Aromatic Amines and Azo Dyes  2-naphtylamine (produces bladder carcinoma)  benzidine (produces bladder carcinoma)  2 - acetylaminofluorene  4- dimethylaminoazobenzene (produces liver tumors) C. Natural Products  aflatoxin B1 (potent hepatocarcinogen produced by mold contamination of food)  mitomycin C D. Other  nitrosamine (can be formed by action of nitrites on foods)  some insecticides (chlordane and others)  some metals (chromium and nickel)  carbon tetrachloride Direct-Acting Carcinogens (DNA-reactive)  Alkylating agents - Anticancer chemotherapeutic drugs (cyclophosphamide, busulfan, chlorambucil) - beta-propiolactone - bis (chloromethyl) ether  Acetylating agents 1 - acetylimidazole Initiation-Promotion Phases of Experimental Carcinogenesis of Mouse Skin I > No tumors I P_P_P_P_P_P > Tumors I P_P_P_P_P_P > Tumors P_P_P_P_P_P_P > No tumors P_P_P_P_P_P_P_ I > No tumors Time >(15 - 70 weeks) I, initiation by single application of subcarcinogen dose of polycyclic hydrocarbon. (benzo-a-pyrene) P, promotion by spaced applications of promoting agent, such as croton oil (phorbol ester). Tumors are skin papillomas and carcinomas.

gagpol

env

src

gag - inner capsid proteins pol - reverse transcriptase protein env - outer envelope proteins src - converts normal chicken cells into their cancerous equivalents (protein - tyrosine kinase) • serine • threonine • phosphatldyl inositol Activation Cancers Protooncogene c - myc of Cellular Proto-oncogenes in Human Associated Change Translocatio n 8 -14, 8 - 2 or 8 - 22 Translocatio n 9 - 22 Cancer Burkitt’s

Chromosom al Activation by Genetic rearrangem ent Genetic rearrangem ent Point mutation Point mutation Gene amplification

c - abl c - H -ras c - K - ras N - myc

Chronic myeloid leukemia Bladder carcinoma Lung and colon carcinoma Neuroblasto ma

Conversion of Cellular Proto-oncogenes to Oncogenes  translocation: c -myc, c-abl  promoter insertion: c -myc, c -erb B  point mutation: c -ras  deletional mutation: c -erb B  amplification: c - myc  Transcription factor (AP-1) complexes with jun: fos  Transcription factor (AP-1) complexes with fos: jus Table: Cellular Functions of Protooncogene Encoded Proteins Protooncogene Associated Function Protein Human Cancer Growth PDGF sis Osteosarcoma factor GrowthEGF Breast, lung, factor receptor erb B ovarian cancer receptor PostGTP – Lung, colon, receptor binding ras pancreatic Class Members cancer transduction protein DNA Viruses Nuclear Breast, colon, Papovirus SV40 transcription myc myc Polyomavirus, cancer, lung virus, papillomavirus regulator protein Burkitt’s Adenovirus Adenoviruses 12, 18, and lymphoma 31 Herpesvirus Epstein-Barr virus, Herpes Simplex Type 2 Virus Hepadnavirus Hepatitis B virus RNA Viruses Retrovirus Murine Sarcoma & Type C Leukemia viruses, Avian sarcoma & Leukemia 3 viruses, Human T cell Leukemia viruses I and II Retrovirus Mouse mammary tumor Type B virus

Some Important Tumor Viruses Oncogenes of Rous Sarcoma Virus

 Retrovirus (RNA tumor virus)
Virus of chicken 9000 nucleotide long

Oncogenes:

Human Genes 9 (q24) Associated with Cancer abl Chronic Susceptibility and Expressions Affected Gene Chromosome c - myc 8 (q24) Burkitt’s Associated Cancer
myeloid leukemia lymphoma ras colon, lung, pancreas, leukemia N -myc small cell lung cancer RET 10(q11) Medullary multiple endocrine thyroid carcinoma, neoplasias 2(p) Neuroblastoma, 12(p) Variety of cancers:

A

B

H chain genes _____....……..

H chain genes

………..

C-MYC

mRNA

mRNA

4. Gene Amplification 5. Point mutation From Harper’s 25th edition: Mechanism of Action of Proto-oncogene to oncogenes: 1. Promoter insertion - certain retroviruses lack oncogenes but may cause cancer over a long period of time - DNA copy (cDNA) of RNA genome is synthesized by reverse transcriptase - cDNA integrated into the host genome (cDNA) known as 'provirus' after integration - cDNA copies are flanked both ends by long terminal repeats - myc gene activated by upstream, adjacent viral long terminal repeat acting promoter - transcription of both myc RNA and translation of the product 2. Enhancer insertion - provirus is inserted dowstream from myc gene 3. Chromosomal translocations - piece of one chromosome is split off and then joined to another chromosome - produces abnormal juxtaposition of BCR gene on chromosome 22 with pare of C-ABL gene on chromosome 9 - this results in chimeric activity BCR-ABL mRNA w/c encodes bcr-able fusion protein with increase protein tyrosine kinase activity - this transforms normal cell to leukemic cell - example: Berkitt's lymphoma 4. Gene amplification - example is methotrexate (inhibitor dihydrofolate reductase), and c-ras - tumor cells becomes resistant to drug of

Mechanisms Involved in Activating PML/RAR-alpha (15;17) Acute Proto-oncogenes to tOncogenes
promyelocytic leukemia
A

1. Promoter insertion
B Provirus LTR LTR

_____

myc …………

myc ……____

myc mRNA 2. Enhancer insertion A myc ……... _____ B
Provirus

myc LTR ………..

LTR

myc mRNA

3. Chromosomal Translocations
8 p 8 8

q p q
14 14

Break

14

5. Point mutation - v-ras oncogene differs from c-ras genes by substitution at position 12 of p 21 - this mutation appears to affect its conformation and diminish its activity as a GTPase
H chain genes

Break

C -MYC

Growth Factor Signaling and Oncogenes

 Growth factors are extracellular proteins that signal cells
to divide, differentiate migrate and die  Interact specifically with high affinity cellular recetors, which span the membrane and have intracellular signaling properties such as tyrosine kinase  Occupancy of receptor triggers an intracellular signaling cascade • Many proonconogenes encode proteins in this signaling machinery

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 Signaling cascade consists of network of interacting
proteins which are: • Receptor tyrosine kinase • Adaptor proteins • Small GTP-binding proteins • Kinases (serine threonine kinases) • Transcription factors ∆ - EGFR: growth factor receptor, deletion mutation • deletion mutation – eliminates exons 2 to 7 in the extracellular domain leading to ligand-dependent activity ras: signaling molecule, point mutation • cellular ras – only active when GTP is bound. It cleaves GTP to GDP + P1, switching itself OFF • v-ras or mutated cellular ras – has lost the ability to hydrolyze GTP and so they activated mos: cytplasmic protein kinase, activation mutation • intracellular protein kinases – found in the MAP kinase cascade can be mutated in such a way that they are constitutively active abl : cytopalsmic protein kinase, fusion • protooncogene is a non-receptor tyrosine kinase is the abl gene • activated by fusion with other proteins following chromosomal breaks • Philadelphia chromosome: an abnormal chromosome t(9:22) resulting in creation of ber-abl fusion protein, with enhanced tyrosine kinase activity conferring growth factor independent growth. This is common in adult – chronic myelogenous leukemia. myc : transcription factor, amplification • transcription factors: myc or jun, can have oncogenic activity by mutation or misexpression • mechanism Is not clear, but it most likely to involve misregulation of downstream genes

 pRb suppresses the entry of cells into the S phase of the
cell cycle  G1 - S transition point

 “two-hit” model  mutational losses of function of both allelic genes at Rb
locus in chromosome 13 q14  proposed by Knudson: - first mutation affecting retinal cells maybe inhibited at birth - second mutation - at the same locus is somatic and random P53 Tumor Suppressor Gene Human chromosome 17 (p13) normal (wild type) nuclear protein, Mw = 53 kdaltons recessive to the normal (wild type) allele in somatic cells “guardian of the genome” “two hits” are required for loss of function normal p53 protein is a transcription factor that mediates: regulation of cell division DNA repair apoptosis  arrest cell cycle at G1 to S transition part

    

DNA Mismatch - Repair Genes  New class of cancer susceptibility genes identified in hereditary non-polyposis colorectal cancer (HNPCC)  A defective hMSH2 gene located in human chromosome 2p and a defective hMLH1 gene located on human chromosome 3p Apoptosis  Greek: apo - away; ptosis - falling  normal process of non-random cell death that occurs in biological conditions: embryological development deletion of self responsive T cells normal cell turnover throughout life genomic DNA damage active, energy dependent process  oncogene - “ accelerator” of cell proliferation or transformation tumor suppressor genes - “brake” apoptosis - “suicidal crash”

Oncogene Co-operativity

 To characterize gene as oncogene – transfect it to
normal fibroblasts and look for the formation of foci (group of dense growing cells)  transformed cells.  This study showed that – often one oncogene was not enough to yield full cellular transformation.  A nuclear and a membrane oncogene was necessary. Ex.: v-ras + c-myc  CANCER = MULTIGENE DISEASE Tumor Suppressor Genes While the activation of some genes may result in cancer, it is reasonable to suppose that activation of other genes may suppress the development of cancer:  Anti-oncogenes  Loss or inactivation of putative tumor suppressing genes may remove a block to cell proliferation and the development of the neoplastic state Retinoblastoma

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 Most common primary malignant eye tumor of children  Normal (mild type) Rb gene on human chromosome
13 (q14) is a transcription regulator

 Protein product pRb has an important functions in the
regulation of the cell cycle and the suppression of tumor cell proliferation

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