O H O Table 2.1 Estimates of the proportion of cancer attributable to speci \201c exposures 0 2 Exposure Estimates (%) ( 3 USA Best estimate USA Nordic countries Men Women Worldwide 1 0 Range ) Tobacco Passive smoking Alcohol consumption Diet Obesity Food additives Sedentary lifestyle Infections Sexual behavior Occupation Perinatal factors/growth Reproductive factors Enviro nmental pollution Industrial products Man-made ionizing radiation Random Solar and ultravio let radiation Medicines and procedures Socio-economic status Geographical factors Family hi story of cancer 30 (25 \200\22340) 15 30 19 <1 2 ? <1 9 <1 2 ? 1 1 1 3 35 <1 (2 \200\2234) (10 \200\22370) ( \200\2235a \200\2232) 3 30 / 1 6 / 30 \200\22340 0 1 5 5 2 1 2b 3b 16 10 1 4 (1 \200\223?) (1) (2 \200\2238) 1 : 0 5 5 3 2 3 <1 6 A 6 2 <1 (0 \200\22312) (1 \200\2235) (<1 \200\2232) M P a g 2 <1 4 2 <1 5 e 2 8 (0.5 \200\2233) 1 3 5 1 1 3 (2 \200\2234) a Allows for protective effect of preservatives b Infection with human papillomavirus or He licobacter pylori ? No precise data available OHO-02(3-10) 11/16/02 11:06 AM Page 9 OTHER EXPOSURES 9 Some pharmaceutical agents (e.g. immunosuppressive agents, anti-neoplastic drugs, and hormo nal preparations) are human carcinogens. 1 Doll, R. and Peto, R. (1981) The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. Journal of the National Cancer Institute 66, 1191 \200\223308. 2 Parkin, D.M., Pisani, P., Lopez, A.D., and Masuyer, E. (1994) At least one in seven cases of cancer is caused by smoking. Global estimates for 1985. International Journal of Cancer 59, 494 \200\223504. 3 Blot, W.J., Li, J.Y., Taylor, P.R., Guo, W., Dawsey, S., Wang, G.Q., et al. (1993) Nutrit ion intervention trials in Linxian, China: supplementation with speci \201c vitamin/minera l combinations, cancer incidence, and disease-speci \201c mortality in the general populat ion. Journal of the National Cancer Institute 85, 1483 \200\22392. OHO-02(3-10) 11/16/02 11:06 AM Page 10 This page intentionally left blank OHO-03(11-36) 11/16/02 10:44 AM Page 11 Chapter 3 Biology of cancer The role of genetics 13 Molecular biology techniques 15 Cell cycle and its regulation 21 Gr owth of cancer 25 Oncogenes and tumor suppressor genes 27 Cytogenetics and cancer 32 OHO-03(11-36) 11/16/02 10:44 AM Page 12 BIOLOGY OF CANCER 12 Molecular biology is an approach to understanding how organisms work by studying the molecu les \200\224in particular nucleic acids that encode genetic information and proteins that p rocess this informa- tion and carry out activities in cells. It is grounded in other scient i \201c \201elds, particularly biochemistry, chemistry, microbiology, genetics, and math /computer science. Molecular biology is a development of biochemistry, with less emphasis on the chemistry of biological molecules and more on how they function within cells and organisms. To study thi s, convenient biological systems are needed and microbiology plays a crucial role. Yeast an d bacteria are often used in experiments because they are simple, cheap, and ethically acce ptable. In cancer, there are two excellent recent examples of this: (cid:2) Understanding o f the cell cycle (work with yeast). (cid:2) Repair of damaged DNA (using the bacterium Esch erichia coli). Although humans and micro-organisms are a long way apart in evo- lutionary terms, their fun damental cellular processes are remarkably similar. OHO-03(11-36) 11/16/02 10:44 AM Page 13 THE ROLE OF GENETICS 13 The role of genetics Molecular biology relies heavily on genetics; traditionally many inter- esting properties o f organisms have been studied via their inheritance, so identifying the genes responsible. Once the genes are found, how they work can be investigated using molecular biology. In hum an genetics, much emphasis has been on what goes wrong \200\224i.e. the genetics of inherit ed disease. The full implications of this for cancer are now becoming clear, since most of these diseases carry a genetic predisposition of some kind. If these predisposing genes are identi \201ed, we have a new handle on some crucial aspects of the disease process; recen tly several cancer susceptibility genes have been isolated. The Human Genome Project (a mul ti-laboratory collaboration to identify the DNA sequence of all the genes in an organism) i s now complete; the task of identifying genes will become routine. Linking these genes with certainty to the risk of diseases such as cancer is still problematic: there are many othe r factors involved (e.g. environmental). The Human Genome Project has shown the DNA sequence for 30 000 genes. Most are novel with n o resemblance to a gene of known function. The amino-acid sequence of the protein product i s pre- dictable, but current methods do not lead to a prediction of the three- dimensional structure of the protein. Once possible, there will still be great dif \201culties in de \201ning the role of novel gene products in the organism \200\231s biology. Applications to health care (cid:2) Using genetically engineered vaccines, therapeutic proteins and hormones, antibodie s, etc. \200\224essentially existing therapies with improved products. Identifying new drug targets and therapeutic strategies. These will come from research into identi \201cation of genetic factors in disease and the development of methods for relating protein sequence (deduced from the DNA sequence) to the function of the protein and its role in pathology. (cid:2) (cid:2) Developing DNA (or derivatives) as drugs i.e. gene therapy. There have been promisi ng preliminary results with diseases such as cystic \201brosis, but there is still a long way to go before this becomes a general therapeutic method. The question of individual ris k modi \201cation according to genotype and attendant issues of privacy, right to know, co nsequences for those with a \201nancial stake in the individual \200\231s health, are imp ortant. Because genetic risk OHO-03(11-36) 11/16/02 10:44 AM Page 14 BIOLOGY OF CANCER 14 factors are already being identi \201ed, these are pressing issues which can only be resol ved by full and informed debate between the public, lawmakers, scientists, and clinicians. OHO-03(11-36) 11/16/02 10:44 AM Page 15 MOLECULAR BIOLOGY TECHNIQUES 15 Molecular biology techniques Scienti \201c advances All cancers arise as a result of changes in genes that regulate cel l growth and behavior; DNA research now offers the greatest potential for the development o f new cancer diagnostics and therapies. Molecular medicine is thus concerned with turning g enes into new therapeutic targets. Developing and applying molecular technologies is the ke y to a new understanding of cancer. Molecular techniques comprise two groups: lysate and analysis and in situ analysis. (cid:2) With lysate methods, tumor biopsies are homogenized and spatial r elationships between tumor cells are destroyed (Southern blot analysis and PCR). This leads to loss of information on hetero- geneity and small subpopulations and an averaging of cha nges. However, quantitation can be simpler and more accurate than in situ approaches. In si tu techniques such as \202uorescence in situ hybridization (FISH) allow the genetic make- up of individual cells within their histological context to be visualized. (cid:2) Genetic analysis Southern blot analysis Loss of genetic material from cancer cells is a classic marker for t he presence of potential tumor suppressor loci. Southern blot analysis, using polymorphisms occurring within the human genome (RFLPs), identi \201es genetic alterations in the cance r cells. It involves homo- genization of tissue samples to extract DNA, followed by cleavag e of DNA with restriction endonucleases. Digested DNA is size-separated by gel electrophore sis and transferred to \201lters for hybridization to labeled probes to provide informati on on sequences of interest. Limitations: (cid:2) Low sensitivity for detection of altered sequences. (cid:2) Large amounts of high-q uality genomic DNA needed. (cid:2) Lengthy procedure. Many of these have been overcome with the use of polymerase chain reaction (PCR) methodologies, but Southern blot analysis remai ns the standard in many situations. OHO-03(11-36) 11/16/02 10:44 AM Page 16 BIOLOGY OF CANCER 16 Table 3.1 Landmarks in molecular oncology 1953 Watson & Crick, molecular structure of DNA. 1972 First recombinant DNA molecule made with ligase (Paul Berg). 1974 Doxorubicin, anti-cancer drug, receives FDA approval. 1975 DNA sequencing developed by Frederick Sanger. 1975 Southern blot technique developed to identify DNA fragments. 1976 SRC, the \201rst human oncogene, discovered. (Now over 50 oncogenes are known.) 1976 Genentech, the \201rst biotechnology \201rm, founded. 1978 Cisplatin and Tamoxifen, anti-cancer drugs, receive FDA approval. 1978 Restriction fragment length polymorphisms (RFLPs) discovered. 1979 p53 gene discovered. 1981 First recombinant vaccine for hepatitus B virus in liver cancer. 1982 Transgenic mouse overexpressing growth hormone. 1985 Polymerase chain reaction (PCR) invented. 1986 RB1 (retinoblastoma gene), the \201rst of over 20 known tumor suppressor genes, cloned. 1990 Human Genome Project launched. 1991 Expressed sequence tags (ESTs) developed. 1991 First human gene therapy for cancer (melanoma). 1992 Paclitaxel, anti-cancer drug, receives FDA approval. 1993 hMSH2, the \201rst of the hereditary non-polyposis colon cancer genes, cloned. 1994/1995 BRCA1/BRCA2, inherited breast cancer genes, cloned. 1996 First eukaryote genome sequenced, Saccharomyces cerevisiae. 1996 Topotecan, anti-cancer drugs, receives FDA approval. 2000 Completion date for sequence of the complete human genome. PCR A huge resource of micro-satellite markers distributed throughout the genome can be use d to map and characterize genetic alterations. Primer pairs \202anking regions of interes t can be used to amplify the region; the size of product reveals information on the genetic change. OHO-03(11-36) 11/16/02 10:44 AM Page 17 MOLECULAR BIOLOGY TECHNIQUES 17 Table 3.2 Tools of the trade Genetic analysis Molecular Cytogenetics Fluorescence in situhybridization Interphase cytogenetics Comparative genomic hybridization Southern blot analysis PCR DNA microchip technology In situhybridization RNA analysis Northern blot analysis RT-PCR Gene expression microarrays Gene expression pro \201ling Differential display RT-PCR Serial analysis of gene expression (SAGE) Suppression subtractive hybridization Immunohistochemistry Protein analysis Western blot analysis ELISA assay Enzyme assays Electrophoretic mobility shift assay DNA footprinting Analysis of gene function Gene transfer into tissue culture cell models Transgenic mice Knockout mice DNA sequence analysis \200\224bioinformatics Database homology searching GeneBank database of DNA sequences BLAST sequence similarity analysis Gene identi \201cation Pattern identi \201cation e.g. transcription factors DNA structure and composition Sequence assembly and contig management Major advantages: (cid:2) Speed of analysis. (cid:2) High-density coverage of the human genome with markers. (cid:2) Automation. (cid:2) Small amounts of sample required. OHO-03(11-36) 11/16/02 10:44 AM Page 18 BIOLOGY OF CANCER 18 Genomic DNA Cut with restriction enzyme Restriction fragments of DNA, length determined by location of recognition sites for restri ction enzyme \200\223 Gel electrophoresis of DNA fragments + Transfer to nitrocellulose filter by Southern Blot technique DNA fragments visualized by hybridization to probe Southern blot analysis. Combined with micro-dissection of \201xed tissue samples, PCR approaches can also study h eterogeneity and separate normal and cancerous areas for independent analysis. Fluorescence in situhybridization (FISH) Molecular cytogenetics is a rapidly evolving \201eld; techniques like comparative genomic hybridization (CGH) and interphase cytoge- net ics are having major impact on tumor genetics. FISH is a sensitive method for analyzing gen etic change in solid tumors, allowing visu- alization of the genetic make-up of individual cells within their histo- logical context. FISH uses chromosome and region-speci \201c pro bes to assess and rapidly copy the number and rearrangements of chromo- OHO-03(11-36) 11/16/02 10:44 AM Page 19 MOLECULAR BIOLOGY TECHNIQUES 19 somes and genes. Genetically abnormal cells are detected by an aberrant hybridization patte rn in the interphase nuclei (interphase cytogenetics). Major advantages (cid:2) Utility in routinely processed pathology specimens. (cid:2) Speed. (cid:2) Non-radi oactive. A disadvantage is the need for cloned probes. CGH resolves this by using tumor DNA as a com plex probe. Thus CGH detects and maps genetic alterations in tumors without the bias of pro be selection. RNA analysis Identi \201cation and quantitation of gene expression Northern blot hybridization and reve rse-transcription polymerase chain reaction (RT-PCR) are commonly used methodologies for de tection and quantitation of gene expression. Northern blot: standard method for determining transcript size and for comparison of messen ger abundance between samples. For Northern blot analysis, RNA is extracted from tumor lysa tes and size-separated by gel electrophoresis. After transfer to \201lters, speci \201c RNA species are detected by probe hybridization. RT-PCR: RNA is \201rst converted into cDNA and speci \201c genes analyzed using gene-spe ci \201c primers in PCR reactions. RT-PCR is extremely sensitive but generally lacks the q uantitation of Northern analysis. A complementary approach is RNA in situ hybridization, id entifying cellular distribution of RNA sequences within a tumor section, allowing study of tumor heterogeneity and subclone development. Gene expression pro \201ling Limitations of Northern blot and RT-PCR: analysis is restrict ed to only a few genes at a time, and sequence must be known in order to gener- ate probes. Each cell may express 10 000 \200\22350 000 genes, so gene expres- sion patterns may be tu mor-speci \201c. It has been dif \201cult to monitor expression patterns of thousands of genes simultaneously until recently. SAGE: Serial Analysis of Gene Expression (SAGE) can be applied in laboratories with PCR and sequencing capabilities. SAGE is based on generations of clones of short sequence tags der ived from mRNA extracted from target tissue. Each individual tag represents a single mRNA, so sequencing of concatenates describes the pattern and abundance of mRNAs in target tissue . It is thus possible to screen several thousand tags in a few weeks. Sequences are screene d against OHO-03(11-36) 11/16/02 10:44 AM Page 20 BIOLOGY OF CANCER 20 existing databases for known genes, and previously uncharacterized sequences are analyzed i n more detail. With SAGE, data are digitized and reusable, so once sequence information is obtained it can be reused and rescreened against any existing or developing genome database . Bioinformatics: Bioinformatics is the use of computers to analyze biological information; a rapidly developing area of cancer research, given the vast amounts of sequence data produc ed by molecular biol- ogy projects. Nucleotide sequences can be used to interrogate public sequence databases for homology and identi \201cation over the internet. They can also be used to identify putative gene regulatory elements and gene features. The establishment of the Cancer Genome Anatomy Project (CGAP) means tumor-speci \201c patterns of gene expressi on are now being held in databases. OHO-03(11-36) 11/16/02 10:44 AM Page 21 CELL CYCLE AND ITS REGULATION 21 Cell cycle and its regulation Cell cycle phases The somatic cell cycle consists of two alternative phases: (cid:2) S phas e, where DNA is replicated. (cid:2) M phase where the cells divide. Separating these are tw o phases where neither DNA replication nor cell division take place: (cid:2) G1 between M a nd S. (cid:2) G2 between S and M. New \201ndings Cell cycle research has led to the identi \201cation of many molecules th at drive and regulate the cell cycle. One important group are proteins called cyclins \200 \224so-named because concentrations rise and fall in a regular pattern during the cell cycl e. This enables them to activate cyclin-dependent kinases (CDKs) whose activity is needed t o propel cells through the cycle. Additional sub-units also regulate CDK activi- ty, includ ing cyclin-inhibitory proteins (CKIs). M G1 G2 Cyclin B/A CDC2 Cyclin D CDK2, 4, 6 Cyclin A CDK2 Cyclin E CDK2 S Basic cell cycle control. OHO-03(11-36) 11/16/02 10:44 AM Page 22 BIOLOGY OF CANCER 22 Cyclin D1,2,3 pp p p CDK4/6 release S-phase genes pRb E2 p16 p Rb-type proteins and the Rb pathway. This complexity ensures cells have precise co-ordination of events that allow DNA duplicati on and subsequent cellular division. In addi- tion, regulation guarantees maintenance of DN A \201delity and protects against a loss of genetic information. Basic cell cycle control is shown in the \201rst \201gure. Research has established many direct links between disruption of cell cycle control and cancer. G1-S transition Enzyme synthesis Transition from G1 to S phase involves assembly and activation of transcri ption factors allowing the synthesis of enzymes required for DNA replication. At the heart of this process are Rb-type proteins and the \200\230Rb pathway \200\231 as seen in the se cond \201gure. The retinoblastoma pro- tein, Rb, and the related p107 and p130 proteins, bind to a number of transcription factors. These activate genes required for progression th rough S-phase, such as DNA polymerases and nucleotide kinases. Progression through G1 requires the phosphorylation of Rb-type proteins, allowing the relea se of transcription factors and subsequent S-phase entry. Phosphorylation of Rb-type protei ns is controlled pri- marily by the cyclin-D-dependent kinases, CDK4 and CDK6. Kinase activ ity is regulated by \202uctuating levels of D cyclins and CKIs from the p16INK4 and kip f amilies, particularly p16 itself. Timing of transition Exactly when a cell embarks on a G1-S transition is tightly controlled to ensure survival, with factors such as cell size, metabolic state, growth factor availab ility, and DNA damage affecting whether a tran- sition takes place. Not all signaling pathw ays involved have been unraveled. However, cyclin-D synthesis and subsequent activation of CDK4/6 has been linked to growth factor availability. It is suggested that cyclin D acts as a cellular sensor with cyclin synthesis allowing cells to cycle in the presence of appropr iate mitogens. OHO-03(11-36) 11/16/02 10:44 AM Page 23 CELL CYCLE AND ITS REGULATION 23 Pathways Superimposed over cyclin/CDK systems are pathways to detect prob- lems such as DNA damage and cell growth inhibition. The most important is the p53 pathway, which plays a ke y role in G1 arrest, apoptosis, and genomic stability. Cells exposed to DNA-damaging agents often become arrested in G1 in a p53-dependent manner. p53 activation increases transcript ion and therefore protein levels of p21, a CKI that blocks cell cycle progression via its a bility to inhibit Cdk enzymes. p53 upregulation does more than induce G1 arrest; in many ca ses programmed cell death or apoptosis is seen. G1-phase arrest and apoptosis are very dive rgent responses; the mechanisms involved in determining which of these dominates is still l argely unknown. Variables such as the extent of DNA damage and levels of p53 may play a rol e. p53 is the most commonly mutated gene in human cancer \200\224not surprising since loss of control of genomic stability is central to cancer development and p53 is a critical factor in monitoring genomic integrity. S phase First steps: the \201rst step in DNA duplication is the formation of initia- tion complexes and it appears that cyclin and CDK pairs, such as cyclin A and CDK2, are involve d. Regulation: maintenance of the replicative state may also be regulated by members of cyc lin/CDK families. CDKs have been shown to phos- phorylate several DNA replication factors i nvolved in elongation including sub-units of the DNA-binding protein, RPA and DNA poly- mer ase. Both are phosphorylated from the onset of S phase and remain so through G2 up to the e nd of M phase. G2-M transition The transition of cells from G2 to mitosis is dependent on the activity of the cylin/CDK complex, CDC2/cyclin B. Phosphorylation by CDC2 has been linked to several ev ents required for mitosis entry, with targets including nuclear lamins and chromatin protei ns. The activity of CDC2 is tightly regulated to ensure mitotic events do not begin until D NA replication is complete. Activation of CDC2 is a result of accumulation of cyclin B occu rring as cells progress through S phase, peaking in late G2. Inactivation of CDC2 occurs by phosphorylation while dephosphorylation is associated with G2-M transition. Delayed transition is commonly observed in G2-phase cells exposed to agents such as radiati on and chemotherapy. The resulting G2 block is thought to allow time to repair DNA damage, while loss of this G2 checkpoint is often observed in cancerous cells. OHO-03(11-36) 11/16/02 10:44 AM Page 24 BIOLOGY OF CANCER 24 Summary Regulation of cell cycle is crucial for maintaining a normal cellular state. Conver sely, perturbation of the cell cycle may occur in all tumor cells. The view of cancer as a disease of the cell cycle is rapidly becoming accepted; the design of novel drugs and gene therapy strate- gies to treat hyperproliferative cancers is now an integral part of med- ic al research. OHO-03(11-36) 11/16/02 10:44 AM Page 25 GROWTH OF CANCER 25 Growth of cancer The basic mode of growth of tumors is exponential, though devia- tions do occur. One cubic centimeter of tumor tissue may contain over 108 neoplastic cells (in addition to many norma l host cells) and their production from an initial transformed cell requires around 30 gene rations of cell multiplication. During this period of active tumor growth the tumor will be below 1 cm3 and undetectable; \201ve further doublings of cell number will give a volume of around 32 cm3; and \201ve more, a volume of over 1000 cm3. Exponential growth from a sin- gle cell thus gives the impression of a long \200\230silent period \200\231 followed by apparently rapid growth. Growth rate of tumors Volume doubling time is the usual measure of tumor growth rate. Doubl ing times range from a few weeks to over a year, with an average of 3 months. Even a 1-mont h doubling time implies that the duration of the \200\230silent period \200\231 between in itiation of a tumor and the appear- ance of symptoms could be a number of years. Cellular basis of tumor growth Three factors determine the growth rate of a tumor: (cid:2) The average cell cycle times. Values for human tumor cells usual- ly range from 1 \200\2235 days. (cid:2) The growth fraction i.e. the proportion of tumor cells that are proliferating. Out- of-cycle cells are common in tumors, leading to growth fractions that are often less than 5 0%. (cid:2) Cell loss. The rate of loss of cells from tumors can be very high, often reaching 9 0% of cell production, and this is the principal rea- son why the volume doubling time is s o much longer than the aver- age cell cycle time. Cell loss occurs mainly by cell death, ei ther as necrosis or apoptosis. Exfoliation and metastasis are other modes of cell loss. Potential doubling time Labeling of proliferating tumor cells with 3H-thymidine or radio- l abeled bromodeoxyuridine allows calculation of the expected tumor growth rate in the absenc e of cell loss. Such potential doubling times are often less than one week. When tumors rec ur following radiation therapy this is often due to rapid tumor cell re-population. OHO-03(11-36) 11/16/02 10:44 AM Page 26 BIOLOGY OF CANCER 26 Therapy implications The kinetics of tumor growth impinge on clinical management in various ways: (cid:2) Time to recurrence following treatment depends very much on the cellular re-population rate. (cid:2) The effectiveness of proliferation-dependent chemotherapeutic agents is limited by out-of-cycle cells, and therefore by a low growth fraction. (cid:2) Surviving tumor cells appear to accelerate after the start of radia- tion or cytoto xic drug treatment. To allow a gap in a course of ther- apy is therefore much more serious than delaying the start of therapy by the same period. (cid:2) Rapid tumor cell re-population provides a rationale for accelerat- ed radiation therapy. OHO-03(11-36) 11/16/02 10:44 AM Page 27 ONCOGENES AND tumor SUPPRESSOR GENES 27 Oncogenes and tumor suppressor genes Gene types Most cancers are monoclonal, arising from a single cell that has accu- mulated k ey mutations leading to uncontrolled cell proliferation. Such mutations can cause gene func tion to be either enhanced (activated) or lost (inactivated). Genes whose function becomes lost or inactivated in carcinogenesis are termed tumor suppres sor genes. Both gene copies must be inactivated before the tumor suppressor function is com pletely lost (i.e. absence of normal protein) so they can be thought of as recessive. Genes whose function becomes enhanced are termed proto- oncogenes and their mutated form is an oncogene. Proto-oncogenes play an essential role in controlling cell proliferation and encoding growth factors, growth factor receptors, signal transducers, cyto- plasmic regulat ors, and transcription factors. Oncogenes generally behave in dominant fashion. Cell mutations DNA mutations occur with a high frequency in mammalian cells (due to radiati on exposure, metabolic accidents, and exposure to environ- mental carcinogens). Exceptional ly ef \201cient DNA repair mechanisms normally ensure that less than 1:1000 accidental bas e changes in DNA causes a mutation. Mutations take a variety of forms: (cid:2) Point mutati ons (substitution of one base pair of a DNA sequence by another). (cid:2) Translocations (gene arrangement due to chromosomal breakage and rejoining). (cid:2) Gene ampli \201cation (multiple copies of a gene). (cid:2) Deletions (loss of gene tic material \200\224from a single base to a whole gene). Oncogenes A large number of oncogenes have been isolated from transformed cells, oncogenic viruses, and human and animal tumors. Muta- tions in ras genes occur in approximately 20% o f human tumors \200\224 K-ras mutations are particularly prevalent in pancreatic cancer (75 \200\22390%). O H O 0 3 Table 3.3 Human oncogenes ( 1 1 Gene Normal function Associated neoplasm Class of mutation 3 6 H-ras Signal transduction (membrane-associated G protein) Melanoma; carcinoma of the colon, lung, and pancreas Point mutations (codons 12, 13, 59 \200\22361). ) 1 K-ras Signal transduction (membrane-associated G protein) Melanoma; carcinoma of the thyroid; acute myelogenous and lymphoblastic leukemia Point mutations (codons 12, 13, 59 \200\22361). 1 / 1 6 / 0 N-ras Signal transduction (membrane-associated G protein) Melanoma; carcinoma of genitourinary tract and thyroid Point mutations (codons 12, 13, 59 \200\22361). 2 1 myc Transcription factor (operates as heterodimer Burkitt \200\231s lymphoma; carcinoma of lung , with protein partner Max) Translocation; (t(8:14) 75% of cases) Ampli \201cation 0 : breast and cervix 4 4 L-myc Transcription factor (operates as heterodimer Carcinoma of lung with protein partner Max) Ampli \201cation A M N-myc Transcription factor (operates as heterodimer Neuroblastoma; small cell lung carcinoma Ampl i \201cation with protein partner Max) P a g erb-B1 Growth factor receptor (EGF) Astrocytoma; squamous cell carcinoma Ampli \201cation e erb-B2(neu) Growth factor receptor (BGF) Adenocarcinoma of stomach, breast, and ovary Ampli \201cation 2 8 Src Signal transduction (non-receptor protein tyrosine kinase) Signal transduction (non-recepto r protein tyrosine kinase) Carcinoma of colon Point mutations; deletions (also altered protein phosphorylation) Rearrangement (t(9:22) \200\224Philadelphia chromosome \200\224bcr-abl fusion protein) Abl Chronic myelogenous leukemia O H O 0 3 ( Table 3.4 Human tumor suppressor genes 1 1 Chromosomal function Normal function Gene Associated neoplasm 3 6 ) p53 17p Transcriptional regulator \200\224promotes DNA repair, apoptosis, and differentiation (acti vates Bax, GADD45, p21, mdm2, Cyclin G; represses Bcl-2, myc, fos, PCNA) \200\224is induced by DNA damage and hypoxia; G1/S checkpoint control gene A p53 relative with similar functi ons but unlike p53 is not produced in response to DNA damage (only one functional copy \200 \224other is inactivated via imprinting) Carcinoma of the breast, lung, pancreas, colon; brain tumors; sarcomas; Li \200\223Fraumeni syndrome 1 1 / 1 6 p73 1p Neuroblastoma / 0 2 1 Rb 13q Nuclear phosphoprotein (phosphorylation pattern regulates availability of E2F transcription factors \200\224important regulators carcinoma of cell cycle) Retinoblastoma; osteosarcoma; small cell lung 0 : 4 4 A WT1 11p Regulator of transcription and RNA splicing (represses IGF-2, Wilm \200\231s tumor PDGF A c hain can activate EGR-1 under certain circumstances) M P Cell cycle regulator (inhibitor of Cdk4/ cyclin \200\224progression through G1) MTS1 (p16) 9p Glioma; melanoma; mesothelioma; carcinomas of lung, bladder, and pancreas a g e BRCA1 17q Transcription factor (p53 coactivator, transactivates expression 45% of familial breast can cers (80 \200\22390% of of p21) 2 combined breast and ovarian cancers 9 BRCA2 13q Transcription factor (interacts with the RAD51 protein i.e. participates in DNA repair; has intrinsic HAT activity i.e. histone acetylation) Approx. 30% of familial breast cancers (14% of combined breast and ovarian cancers) APC 5q Forms complex with a- and b-catenin and tubulin Colorectal carcinoma (familial and sporadic) O H O 0 3 ( 1 1 3 6 ) 1 1 / 1 Table 3.4 (continued) 6 / 0 Chromosomal function Normal function Gene Associated neoplasm 2 DCC 18q Cell adhesion molecule (receptor for netrin-1 \200\224axonal chemoattractant) Colorectal carcinoma 1 0 : 4 DPC4 nm23 18q 17q Involved in TGFb-induced growth suppression Nucleoside diphosphate kinase (involved with pr oliferation, differentiation, motility (microtubule assembly and disassembly), and developm ent. Carcinoma of the pancreas Loss of gene is associated with increased metastatic ability; bre ast and colorectal carcinoma 4 A M P a g e 3 0 OHO-03(11-36) 11/16/02 10:44 AM Page 31 ONCOGENES AND tumor SUPPRESSOR GENES 31 Tumor suppressor genes Examination of familial cancer syndromes (e.g. retinoblastoma) and e xperimental evidence (particularly from somatic cell hybrid experi- ments) has demonstrated the existence of a different class of cancer gene \200\224tumor suppressor genes. p53 is t he most frequently-altered gene in human tumors. Approximately 37% of all cancers have a p5 3 mutation (incidence is much higher with cancers of the lung and colon). Certain speci \201c tumor mutations are associated with partic- ular carcinogens e.g. hepatocellular carc inoma is linked with hepatitis B and a \202atoxins correlates with a high incidence of mut ations in codon 249 of p53. Products of tumor suppressor genes have a variety of functions within the cell. A large num ber of genes have now been isolated, as can be seen in the table. Multi-step carcinogenesis The development of cancer is a multi-step process characterized b y repeated cellular insults resulting in the accumulation of mutations. The steps involved in the development of colorectal cancer have been particularly well characterized (see \201gure). Single mutations (e.g. APC) can lead to benign cellular proliferation (familial polyposis coli) that predisposes to the development of malignancy. Mutations in DNA repair genes (e.g. MMR \200\224mis-match repair) speed up this process of mutation accumulation. p53 Other changes ? DCC DPC4 JV18 ? APC DNA hypomethylation K-ras Early adenoma Late adenoma Intermediate adenoma Normal epithelium Carcinoma Metastasis Dysplastic MMR deficiency Genetic alterations associated with colorectal carcinogenesis. Mutation frequencies in MMR- de \201cient cells are two to three times higher than in normal cells \200\224such MMR mut ations are found in > 70% of hereditary non-polyposis colorectal cancer (HNPCC) cases and > 65% of sporadic colorectal cancers exhibiting micro-satellite instability.These cases acco unt for 15 \200\22317% of total colorectal cancers. (Adapted from Kinzler and Vogelstein). OHO-03(11-36) 11/16/02 10:44 AM Page 32 BIOLOGY OF CANCER 32 Cytogenetics and cancer Cytogenetic analysis in cancer is used to con \201rm diagnosis, to aid differential diagno sis, and (possibly) to indicate prognosis. Nowell and Hungerford \201rst described a speci \201c chromosome abnor- mality associate d with chronic myeloid leukemia in 1960. Since then, numbers of chromosomal abnormalities a ssociated with cancer have grown exponentially. Abnormalities can be structural, numerical, or both; they may be restricted in distribution or found in many different tumor types. So me chromosomal alterations are useful in the diagnosis and prognosis of malignancies; the s igni \201cance of others is unknown. Nomenclature Last revised in 1995, cytogenetic abnormalities are described by the ISCN (Int ernational System for Human Cytogenetic Nomenclature). Summary of the nomenclature used: + or \200\223 Gain or loss of chromosome following the symbol. p q del Short arm of chromosome. Long arm of chromosome. Deletion. A deletion in the chromosome dis tal to breakpoint, or between two breakpoints if two are stated. Derivative. A chromosome d erived from a rearrangement. Rearrangement (if known) is described. Dicentric. Chromosome h as two centromeres derived from the two named chromosomes. der dic dup Duplication. A segment of chromosome de \201ned by two break- points is duplicated. Isochromosome. Chromosome is composed of two identical arms rather th an a p and q arm. Insertion. Segment from one chromosome is inserted into another. Inversio n. Segment of chromosome de \201ned by two break- points is inverted compared to its norma l orientation. Translocation. Swapping of segments distal to the stated breakpoints in name d chromosomes. i ins inv t Chromosomal abnormalities structural (cid:2) numerical Examples include: (cid:2) Chronic myeloid leukemia (cid:2) \200\224small chromosome 22 (Philadelphia chromosome) OHO-03(11-36) 11/16/02 10:44 AM Page 33 CYTOGENETICS AND CANCER 33 (cid:2) Acute myeloid leukemia \200\224many cytogenic abnormalities (80% of patients) (cid:2) Therapy-related AML (cid:2) Myelodysplastic syndromes \200\22450% have chromosomal abnormalities (cid:2) Myeloproliferative disorders (cid:2) Acute lymphocytic leukemia \200\22480% have chromosomal abnormalities (cid:2) Chronic lymphoproliferative disorders (cid:2) Lymphoma \200\224many structural and numerical chromosomal abnormalities (cid:2) Neuroblastoma \200\224deletion of 1p and ampli \201cation of N-myc (cid:2) Wilms \200\231 tumor (nephroblastoma) \200\224many chromosomal abnormalities reported Invasion methodology Tumor invasion and metastases is a complex, dynamic, multi-step proces s: Initial invasion of tumor through basement membrane. (cid:2) Movement into connective tissu e surrounding tumor cells. (cid:2) Invasion of tumor cells into blood vessels. (cid:2) (cid:2) Circulating tumor cells are arrested in blood vessels of a distant organ or tissue; tumor cells invade organ from blood vessels. (cid:2) Tumor cells then grow within tissue to form a metastatic tumor that may become clinically evident. (cid:2) Process of tumor invasion and metastases results from alterations in cell-to-cell a nd cell-to-matrix adhesion and increased matrix degradation. Extracellular matrix degradation Several stages during the process of tumor invasion and me tastases require increased degradation or breakdown of extracellular matrix or connective t issue surrounding tumor cells. The extracellular matrix is a complex mixture of proteins in cluding different types of collagen, elastin, \201bronectin, and laminin. Digestion of ex tracellular matrix is carried out by several groups of proteolytic enzymes. Major groups of enzymes implicated in tumor invasion and metastases are: Matrix metalloproteinases (MMPs) A family of zinc-containing enzymes involved in the degrad ation of the extracellular matrix; considerable evidence indicates that OHO-03(11-36) 11/16/02 10:44 AM Page 34 BIOLOGY OF CANCER 34 individual MMPs have an important role in tumor invasion and tumor spread while expression of individual MMPs in tumors may be associated with prognosis2. MMPs are broadly classi \201ed into collagenases, gelatinases, stromelysins, and the recently-identi \201ed membra ne-type MMPs. MMPs are secreted proteins, produced as pro-enzymes and activated by cleavage of a N-termin al propeptide. Gelatinases, particularly MMP-2, appear to be important in initial stages of tumor invasion as they degrade components of the basement membrane, while other MMPs contr ibute to later stages of tumor invasion. Membrane-type MMPs that are membrane-bound appear to be involved in activation of MMP-2, which is capable of activating other MMPs. Activity of MMPs is regulated by interaction with naturally occurring inhibitors, tissue in hibitors of metalloproteinases. Clinical interest in MMPs is considerable since the use of synthetic, low- molecular-weight, broad-spectrum inhibitors of MMPs can prevent tumor sprea d in human tumor xenografts. Several MMP inhibitors are being developed for clinical use. Plasmin system Urokinase-type plasminogen activator (uPA) is a serine protease that catalyz es the activation of plasminogen to plasmin \200\224a broad-spec- trum protease that in tur n can break down a variety of extracellular matrix components. Plasmin can also promote the activation of MMP-2, thus linking plasmin system and MMPs in tumor invasion. Cathepsins A group of lysosomal proteolytic enzymes that also degrade many com- ponents of the extracellular matrix. Widely expressed in tumor cells, stromal cells, and endothelial c ells. Cathepsins can be activated by uPA. Cell adhesion Tumor invasion and metastasis is also characterized by alterations in both ce ll-to-cell and cell-to-matrix adhesion. Cellular adhesion both to adjacent cells and surrou nding extracellular matrix is mediated by a variety of molecules including: Cadherins Transmembrane glycoproteins involved in cell adenomatous polypo- sis coli (APC) g ene product. The most important cadherin in relation to tumor invasion is E-cadherin whose expression is downregulated in various types of malignant tumor; loss of E-cadherin frequen tly appears to correlate with tumor invasion and metastasis. Integrins Transmembrane proteins involved in cell to matrix adhesion. Individual integrins are receptors for a variety of matrix proteins OHO-03(11-36) 11/16/02 10:44 AM Page 35 CYTOGENETICS AND CANCER 35