[page 21↓]


A malignant cancer cell is characterized by autonomous and invasive growth as well as the capability to metastasize to distant sites of the body and to infiltrate blood and lymph vessels. The pathway to tumor formation is a multi-step journey, it takes genetic alterations in at least four pathways to convert a normal cell to a cancerous cell and to evoke derangement of numerous gene products (Weitzman and Yaniv 1999). The phenotype might change with every new genetic event succeeding, e.g. normal epithelia progresses from dysplasia to adenoma to carcinoma in situ to invasive carcinoma while it aquires additional genetic aberrations (Vogelstein and Kinzler 1993). Cells are quite resistant to neoplastic formation by a number of intrinsic mechanisms controlling the cell cycle and they might compensate loss of function in one pathway with gain of function in another. Loss of normal growth control is due to mutation in three categories of genes: Proto-oncogenes, tumor suppressor genes and DNA repair enzymes. All cells are able to replicate themselves but will eventually be bound to reach a non-dividing state, the so-called senescence. Genetic alterations, however, may make it possible for the cell to escape this state. After living through a crisis and usually massive cell death the cell may become immortalized and duplicate eternally, without growth factors and anchorage to a solid ground.

4.1. The cell cycle

The mechanism of cell division is necessary for the understanding of transformation in neoplasia. The cell cycle is subdivided into G (gap) 1 phase, in which the cell decides whether or not to continue to S phase, S (synthesis) phase, during which the genome is replicated, G2 phase, where replication errors are detected and corrected and M (mitosis) phase, which gives room to separation of the replicated chromosomes and packaging them into two new nuclei as well as division of the cytoplasm (cytokinesis). Replication errors occurring during S phase are corrected by the cell. Various mechanisms are available and mismatch repair is essential for preventing mistakes to be passed on to the daughter generation. Numerous cancer types display a defect mismatch repair system and are strikingly involved in neogenesis (Eshleman and Markowitz 1996). Telomere biology also is another key word in neoplastic events. Telomeres are the structures at the end of our chromosomes. With each cell cycle the telomeric DNA is left somewhat shorter and this telomeric erosion is thought to be one of the restricting factor to the cell´s lifespan. Normal cells naturally have low telomerase activity, the RNA-dependent DNA polymerase that replicates telomeric DNA and holds the responsibility for keeping telomere length, while cancer cells express telomerase at higher levels and thus escape normal lifespan control (Weitzman and Yaniv 1999). Transition between G1 and S and G2 and M is subjected to strict control by checkpoints, and checkpoint regulation mechanisms imply two cyclin-dependent kinase (cdk)/cyclin complexes: cyclinD/cdk4 or -6 and cyclinE/cdk2. Activation of cdk/cyclin complexes initiates transcription of factors enhancing growth and differentiation, whereas inhibition of cdk/cyclin complexes by cdk inhibitors (cdki) leads to cell cycle arrest or apoptosis, e.g. p21, which upregulates the p53 gene product upon detection of DNA damage (Gartel, Serfas et al. 1996). Apoptosis is a form of programmed cell death, functioning as the regulatory mechanism in cell homeostasis opposite to mitosis. Signals from the extra- or intracellular space, e.g. tumor necrosis factor (TNF) binding to its receptor or p53 (upgraded by vast DNA damage and subsequent pathways) initiate activity of the interleukin 1 (IL1)-converting enzyme (ICE) family of proteases and result in DNA degradation on the nuclear level and successively, cell death (Carson and Lois 1995; Martin and Green 1995; Ledgerwood, Pober et al. 1999).

4.2. Cancer genes

Tumor formation is thought to be induced by cooperation of mutations causing telomerase upregulation and mutations in proto-oncogenes or tumor suppressor genes.

Proto-oncogenes are normal genes controlling cell growth and are contained in normal cells. They might, however, be transformed and activated to oncogenes, by point mutation, amplifi-cation or chromosomal rearrangements. About 100 such oncogenes have been identified so far and are activated in numerous human cancers, e.g. ret, the gene for a receptor tyrosine kinase which displays mutations in MEN2A and FMTC (familial medullary thyroid carcinoma) (Calender 1998).

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Table 1. Examples of oncogenes




Growth factor


Tyrosine kinase


Receptor lacking proteine kinase activity


Membrane-associated G protein


Cytoplasmic protein serine kinases


Cytoplasmic regulator


Cell cycle regulator


Transcription factors


Transcription elongation factors


Intracellular membrane factor




Adapter protein


RNA binding protein

Tumor suppressor genes (TSGs) are mutated genes found in the majority of human neoplasms (Weinberg 1991).Analysis of retinoblastoma cases lead to the emergence of Knudson´s two-hit hypothesis about carcinoneogenesis (Knudson, Di Ferrante et al. 1971). A TSG is typified by the very critical function of suppressing uncontrolled cell growth and to enhance cell differentiation. A TSG being the cause of familiar cancer types presents with the following features: One allele generally experiences loss of gene function by a germline mutation of the respective allele and a somatic mutation leads to loss of the second wildtype allele (Marshall 1991). The retinoblastoma gene (Rb) and the gene causing familiar adenomatosis (APC) are examples for TSGs. Analysis of retinoblastoma cases lead to the emergence of Knudson´s two-hit hypothesis about carcinoneogenesis (Knudson, Di Ferrante et al. 1971).

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Table 2. Examples of tumor suppressor genes



associated tumors


cell cycle regulator, promotes

growth arrest and apoptosis

most sarcomas, breast

carcinoma, leukaemia


binds alpha-and beta-catenin:

may mediate adhesion, cell

cycle progression

Colon carcinoma



Ca2+pendent intercellular

adhesion, signalling

many: Breast, ovarian


modulates RNA polymerase-II

via elongin

Renal cell carcinoma,



DNA mismatch repair

Hereditary non-polyposis

colon cancer


cell growth inhibitor

Pancreas, colon

4.3. Knudson´s two-hit hypothesis about neoplasia

Two sequential mutations in a neoplasm precursor cell can result in the development of neoplasia. Each cell can be hit postzygotically by a first mutation of a TSG in the germline cell (all cells are mutated identically, hereditary neoplasia) or, in the somatic cell as a more rare event (non-hereditary neoplasia). This usually is a small mutation such as a point mutation and does not result in detectable biological effects on the cell but in a heterogenous carrier predisposed to neoplastic process. A second somatic mutation (=hit) of the remaining wildtype allele of a tumor suppressor gene results in loss of function of the gene and evokes neogenesis. This second hit is more likely to occur early when the first hit is a germline mutation compared to somatic mutations (Knudson 1978). Both mutational events finally lead to uncontrolled cell growth resulting in a tumor clone (Marx, Agarwal et al. 1999).

LOH analysis of retinoblastoma cases leads to findings of somatic hits in retinoblastoma as chromosomal deletion or loss of a whole chromosome (Cavenee, Dryja et al. 1983; Cavenee, Hansen et al. 1985). The second hit can occur as e.g. point mutation, somatic recombination or chromosomal deletion (Knudson 1978). TSGs may be silenced by still another process: hypermethylation of regions promoting gene regulation (CpG islands) thus resulting in inhibition of transcription of that gene. Examples for genes hit by hypermethylation are i.g. p16 and Rb (Schmutte and Jones 1998).

4.4. DNA repair

6 billion base pairs of DNA are copied in each cell division. The DNA can suffer various kinds of damage and the proof-reading activity of DNA polymerase has a certain error rate, it is therefore crucial to have DNA repair mechanisms to grant propagation of the correct DNA sequence to the next generation. There are several DNA repair mechanisms acting upon different types of DNA damage, e.g. DNA mismatch repair and nucleotide excision repair (Kolodner 1996).

These repair systems can be damaged by both acquired and inherited mutations, thus initiating accumulation of genome-wide molecular alterations under cell division. Deficient repair systems causing alterations in TSGs or oncogenes may lead to canceroneogenesis.

The DNA mismatch repair systems hold the function of detecting errors in recently synthesized DNA, attaching to the defective base pairs and excising them. Re-synthesis of the gap and re-ligation by an [page 24↓]enzyme complex terminate the process. MutS and MutL are mismatch repair proteins, first found in procaryotes, with five known human MutS homologues (hMSH2-6) and three MutL homologues (hMLH1, hPMS1 and hPMS2) (Fishel and Kolodner 1995; Fishel 1998). Germline mutations in hMSH2 and hMLH1, e. g. are detected in 90% of hereditary non-polyposis colorectal cancers (HNPCC) (Liu, Parsons et al. 1994) and LOH on the hMLH1 and hMSH3 loci has been found in non-small lung cell cancer (Benachenhou, Guiral et al. 1998). Mutations in mismatch repair genes are associated with a generally increasing mutation rate and cancerogenesis as well as microsatellite length instability. Microsatellites (or simple repeated sequences, SRSs) are up to 6 bp long DNA sequences repeated 10-50 times. They are characterized by relatively low inherent mutation rate, and individual variability.

The nucleotide excision repair system repairs DNA damaged by ultraviolet radiation (UV). Global genomic repair and transcription-coupled repair cut the DNA on both sides of the lesion, re-synthesize the correct sequence and ligate it back into the gap. p53 plays an important role in activation of the global genomic repair pathway and activation is mediated through p48 transcription (Ford and Hanawalt 1997).

Several human cancers have manifested defects of global genomic repair and transcription-coupled repair, among those e.g. various types of Xeroderma pigmentosum, a formof skin cancer caused by exposure to UV (Lambert, Kuo et al. 1995; Chu and Mayne 1996).

4.5. Definition of LOH - Expression of a tumor suppressor gene

The second, carcinogenic insult on a neoplasm precursor cell usually results in removal of the normal copy of the mutated gene, often as large deletions or the whole remaining chromosomal copy. Occasionally, a replacement of the lost DNA might happen by so-called gene conversion,a procedure by which lost DNA is replaced with the respective sequence from the other copy. Heterozygosity (i.e. two distinct alleles in germline DNA) is the essential requisite for evaluation of LOH. If a heterozygous individual loses one allele of a polymorphic site this can be detected as allelic loss or loss of heterozygosity (LOH). The germline DNA remains heterozygous at that site. Thus, screening of LOH has been taken advantage of to narrow and identify regions with putative tumor suppressor genes.

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