Rb In Cervical Cancr

Transcriptional repressor

tion by changing the conformation of proteins and activating enzyme functions; phosphoryla-tion also influences protein-protein interactions. Ras oncogenes were among the first oncogene products implicated in human cancers; at least one third of tumors contain mutated Ras genes. Proteins such as Ras become activated when they bind guanosine triphosphate (GTP) and inactivated when they hydrolyze GTP to guanosine diphosphate (GDP). In the activated state, Ras proteins switch on a cascade of other kinases, which in turn activate the transcription factors Fos and Jun committing the cell to DNA replication and division. Specific mutations in Ras result in the permanently activated state. Ras mutations in rodent tumors induced by chemical carcinogens have been well characterized and drive cell division [34]. Similar mutations are found in human lung, colon and pancreatic tumors [26,35,36].

While altered oncogenes result in enhanced cell proliferation, tumor suppressor genes block tumor growth. When they are inactivated, growth becomes constitutive, no longer subject to normal growth control. Several tumor suppressor genes have been identified and linked to heritable tumors. The first to be identified was the RB gene associated with retinoblas-toma. Inactivation of this gene has also been associated with sarcomas, small cell lung carcinomas, and breast and bladder cancers [37]. Another example is the hereditary mutations which occur in the p53 tumor suppressor gene in Li-Fraumeni syndrome patients in whom cancers in multiple tissues are found [37]. P53 is also the most frequently mutated tumor suppressor gene in sporadic tumors. This may be related to its complex role in many central cellular processes, including gene transcription, DNA repair, cell cycling, genomic stability, chromosomal segregation, senescence, and apoptosis [38]. In addition, several oncogenic DNA viruses mediate their effects, in part, by targeting p53 protein for binding. Tumor suppressor genes are inactivated by "two hits", two successive genetic events which fully eliminate activity of both alleles of the gene. In familial cases, the first "hit" has already occurred in the germline of the patient, but in nonfamilial cases, one hit is a point mutation or small deletion, and the second hit is loss of heterozygosity (LOH). Mapping sites of LOH has been a major route by which new tumor suppressor genes have been identified. While several tumor suppressor genes, such as p53 and RB, are found in both familial and sporadic cancers, others appear exclusively in familial cancers (e.g., BRCA1, WT1). Finally, epigenetic mechanisms, which do not involve alterations in DNA sequence, are also important in tumor suppressor gene inactivation through the process of DNA methylation. Extensive methylation of cytosine at CpGs in regulatory sequences such as promoters can turn off gene expression.

It is apparent that a given tumor can contain multiple types of mutations, and that there is considerable heterogeneity between tumors of the same histologic type, in terms of the types of mutations seen. However, a model has been suggested in which six essential alterations in the cell are required for malignant growth. These include: self-sufficiency in growth signals, insensitivity to growth-inhibitor (antigrowth) signals, evasion of programmed cell death (apoptosis), limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis [3]. It may be necessary in all types of tumors for these capabilities to be acquired, although the order in which they arise may vary, and the specific genes which are responsible for the acquisition of the particular capability may differ. The loss of genomic stability results in increased rates of mutations that drive the accumulation of the changes needed for tumor development. Loss of genomic stability is also responsible for tumor heterogeneity; that is, no two tumors are exactly alike, and no tumor is composed of genetically identical cells [39].

Biological Markers in Carcinogenesis

A variety of highly sensitive and specific laboratory procedures are now available for use as biomarkers to identify individuals exposed to particular chemical carcinogens, to determine individual risk for cancer development, to identify individuals with early-stage clinical disease, and also to use as intermediate endpoints in intervention studies. Figure 1.2 provides a schematic diagram of the sequence of events in the continuum from the initial exposure of an individual to a causative agent(s) to the eventual development of a fully malignant tumor. Several types of biomarkers make it possible to precisely monitor each of these events [40,41]. These biomarkers can be divided into specific categories: internal dose, biologically effective dose, early biologic effects (responses), and susceptibility. Biomarkers of internal dose take into account individual differences in absorption or bioaccumulation of the compound in question and indicate the actual level of the compound within the body and in specific tissues or compartments. Examples include measurement of cotinine in serum or urine, resulting from cigarette smoke exposure; measurement of 1-hydroxypyrene in urine from PAH exposure; measurement of aflatoxins in urine from dietary exposure; and measurement of organochlorines in serum from dietary exposure. These biomarkers occur early in the continuum from exposure to disease, and therefore, while they are good markers of exposure, they are generally not useful in the identification of risk or early stage disease.

Another major area of research has been the identification of genetic susceptibility factors specifically related to carcinogen metabolism

Cancer Markers Numbers
Figure 1.2. Biologic markers in chemical carcinogenesis.

and DNA repair. Phenotyping assays have been used in a number of studies to investigate relationships between enzyme activity and cancer risk. For example, a lack of activity of GST M1 has been associated with increased risk of lung cancer in a number of studies [reviewed in [42]]. Deletion of this gene can also be determined using polymerase chain reaction (PCR) methods, simplifying the investigation of the role of this enzyme. Similarly, single-nucleotide polymorphisms in a number of the phase I and II enzymes responsible for the activation and detoxification of chemical carcinogens have been identified [43]. These polymorphisms are frequently associated with altered enzyme level or function. Thus, a major area of research has been the determination of the frequency of specific polymorphisms in individuals with or without various cancers. A number of studies have demonstrated elevated risk for cancer development among carriers of specific alleles. While the increase in risk is often small (<2-fold) compared to that in carriers of germline mutations in specific tumor suppressor genes such as p53 or Rb, the high prevalence of these alleles suggests that they are associated with a high attributable risk in the population. The recent discovery of polymorphisms in DNA repair genes have further expanded these studies. A number of published reviews summarize this area of research in detail [42-45].

Biomarkers of Biologically Effective Dose

These assays measure the amount of the compound that has actually reacted with DNA or with an established surrogate target, for example proteins in the blood. Assays for carcinogen-DNA adducts in the target tissue provide a more relevant marker than assays of the internal dose. This is because the former takes into account not only individual differences in absorption and distribution, but also differences in metabolism (activation versus detoxification) of the chemical and differences in the extent of repair of adducts. Unfortunately, for many studies in humans, DNA from the target tissue is not readily accessible, and thus surrogate tissues are often used (e.g., placenta, peripheral blood cells, buccal cells). The relationship between the types and levels of adducts in these more readily sampled sources and those in the target tissue have been established in some animal studies, but there are few similar studies in humans. A relationship between lung and blood DNA adducts has been observed [46,47]. The time frame of exposure, which can be monitored by measuring DNA adducts, is limited by cell turnover and DNA repair, and thus generally reflects recent exposure. Studies in former smokers have demonstrated that although lung tissue adducts are lost within several years of smoking cessa tion, blood adducts are lost within several months, and oral cell adducts within weeks [48-50].

Measurement of Carcinogen-DNA Adducts

Several highly sensitive and specific methods have been developed for detecting carcinogen-DNA adducts in humans, including physical methods such as fluorescence spectroscopy and gas-chromatography/mass-spectrometry (GC/MS), 32P-postlabeling, immunoassays employing antisera to specific adducts, and combinations of these methods [reviewed in [12,51-53]]. These assays are highly sensitive (able to detect one adduct/107 to 109 nucleotides), but often require an appreciable amount of DNA (about 10-100 mg) and are fairly laborious. Immunoassays require the generation of antibodies, which frequently have cross-reactivity with structurally related adducts. However, the antibodies can be used for immunohistochemical detection of adducts in tissue biopsies or exfoliated cells. The 32P-postlabeling method can detect multiple types of adducts resulting from exposure to complex mixtures; absolute quantitation is a problem, especially with poorly characterized adducts, since the efficiency and yield at each of the multiple steps cannot be easily determined. Other methods of adduct detection include high performance liquid chromatography with fluorescence (aflatoxin B1 and BP) or electrochemical detection (oxidized bases), and GC/MS (alky-lation adducts). These last two methods require the hydrolysis of DNA before analysis.

Carcinogen-DNA adducts have been detected and quantified in individuals exposed to carcinogens in various occupational or environmental settings. Increases in PAH-DNA have been found in foundry, aluminum-plant, and coke-oven workers' and in roofers, fire fighters, coal-tar-treated psoriasis patients, smokers, consumers of charbroiled foods, and subjects exposed to high levels of air pollution. These studies have generally observed consistent increases in DNA damage with increased exposure, but have also revealed large interindividual differences in adduct levels, even in individuals with apparently comparable exposure. These differences are believed to be due to genetic differences in carcinogen metabolism and DNA repair, as well as differences in exposures to other substances (such as the intake of antioxidant vitamins), that may influence carcinogen metabolism.

Measurement of Carcinogen-Protein Adducts

Since the activated metabolites of several carcinogens can also form covalent adducts with proteins, assays of protein adducts have been used as a marker of carcinogen exposure and activation, thus serving as a convenient surrogate for DNA adducts [Reviewed in [12,52,54]]. Although proteins are not critical targets during carcinogenesis, the extent to which they are modified can be a useful biomarker of the biologically effective dose. Because of their abundance in blood, both hemoglobin (extracted from red blood cells) and serum albumin have been used for such assays. As with DNA adducts, protein adducts provide information on only relatively recent exposure, since the life span of the red blood cell is about four months, and the half life of serum albumin is about 21 days. However, protein adducts are not removed by any repair system. Therefore they may be a more sensitive marker of chronic exposure. All of the current assays for protein adducts require the isolation of the modified amino acid or the released carcinogen residues, which is followed by the analysis of these residues. GC/MS has been used to measure ethylene oxide-, 4-aminobiphenyl-and tobacco-specific nitrosamine-hemoglobin adducts resulting from occupational exposure or smoking [Reviewed in [12,54]]. Immunoas-says have been used for quantitation of afla-toxin-albumin [52].

Adducts as Markers of Cancer Risk

While most of the studies on adducts have examined the relationship between exposure and adduct formation, a small number of studies have examined the relationship between adducts and cancer. Two types of studies have been carried out (Table 1.2): case-

Table 1.2. DNA and protein adducts in case-control and nested case-control studies (reference numbers in brackets). Case-Control

PAH-DNA and lung cancer [46,55,56] 4-ABP-DNA and liver cancer [58] Aflatoxin B1-DNA and liver cancer [57]

Nested case-control

Aflatoxin B1-guanine in urine and liver cancer [59,60] Aflatoxin B1-albumin in blood and liver cancer [61-63] Aflatoxin metabolites in urine and liver cancer [64]

control studies, in which blood or tissue samples are collected from cases at the time of diagnosis; or nested case-control studies, in which samples are banked from healthy individuals, and cases are identified as the cohort is followed. The latter type of study is considered ideal since it eliminates the potential problem of the disease influencing the biomarker. In case-control studies, PAH-DNA adducts have been found to be elevated in white-blood-cell DNA of lung cancer cases compared to controls, adjusting for level of smoking [46,55,56], and aflatoxin- and 4-aminobiphenyl-DNA adducts have been found to be higher in liver tissue of hepatocellular carcinoma patients compared to tissue obtained from surgical controls [57,58]. In nested case-control studies, urinary aflatoxin-guanine adducts, aflatoxin metabolites, and albumin adducts have been found to be increased in liver cancer cases, and a synergistic interaction between chronic HBV infection and aflatoxin on liver cancer risk has also been observed [Reviewed in [52]].

Markers of Early Biologic Response Cytogenetic Assays

These include assays for sister chromatid exchange (SCE), micronuclei (MN) and chromosomal aberrations (CAs). As with the adduct studies, these assays have been used most extensively for detection of exposure, but they are not chemical- or exposure-specific. CAs in peripheral lymphocytes have been used extensively as sensitive monitors of radiation exposure, but appear to be less sensitive for detecting exposure to chemical carcinogens [Reviewed in [65]]. MN consist of small amounts of DNA in the cytoplasm which are not incorporated into daughter nuclei during mitosis because of chromosomal damage. MN are easy to score but reflect only a small proportion of the induced chromosome aberrations. A major advantage of assaying for MN is that this assay can also be carried out directly on exfoliated cells (such as oral mucosa cells) which can be sampled noninvasively. Assays for MN have been used to examine the toxic effects of cigarette smoking, and also the beneficial effects of b-carotene, in oral mucosal cells of betel-nut chewers [Reviewed in [66]]. SCEs in peripheral blood lymphocytes are considered a more sensitive, rapid and simple cytogenetic endpoint than CA for evaluation exposure to genotoxic agents. Increased levels of SCEs have been demonstrated as a result of exposure to cigarette smoke, certain occupational exposures, dietary factors, and certain drugs [Reviewed in [67]].

Cytogenetic assays have also been used in nested case-control studies to determine whether they are predictive of risk. Combined analyses of data from Nordic and Italian prospective cohort studies found that chromosomal aberrations were significant predictors of risk for all cancers and were independent of age at test, gender, and time since test; the relationship was not affected by the inclusion of occupational exposure level and smoking habit [68,69]. No association was found between MN or SCE and subsequent cancer incidence/mortality.

Acquired Gene Mutations in Oncogenes or Tumor Suppressor Genes

Numerous studies have investigated mutations in oncogenes and tumor-suppressor genes in tumors of both animals and humans. The p53 tumor suppressor gene is probably the most extensively studied gene and is mutated in about 50% of various human tumors [reviewed in [70,71]]. This work has also demonstrated links between environmental exposures and specific mutational spectra present in the tumor. These links include a relationship between aflatoxin B1 exposure and codon 249 mutations in liver cancer, sunlight and mutations that are characteristic of those produced by UV-induced pyrimidine dimers in skin cancer, and G-T transversions, characteristic of bulky carcinogens, in cigarette smoke and lung cancer [72]. Similarly, studies of mouse skin, rat mammary tumors, and mouse liver tumors indicate that the types of base-substitution mutations seen in ras genes in these tumors depend on the specific carcinogen administered, and that these types of mutations often correlate with the type of DNA base modification and the mutational spectra of these chemicals in simpler systems [36].

While these studies have been very informative for understanding disease etiology, newer markers involving the use of blood, urine, and sputum to identify circulating tumor cells and DNA and oncogene and tumor suppressor gene proteins are more likely to be useful precursors to clinically detectable disease (Table 1.3). Blood levels of mutant oncogene and tumor suppressor gene proteins have been measured using Western blot and enzyme-linked immunosorbent assay (ELISA) technology. In one study, a subset of patients with mutations in the p53 gene in their tumor carried mutant p53 protein in their serum [73]. However, in another study [74], p53 was only moderately increased in serum of lung cancer patients

Table 1.3. Examples of biomarkers for early stage disease (reference numbers in brackets). Proteins in Plasma/Serum

Mutant p53 and ras [74,75,77] Overexpression of c-erbB-2 [80] Antibodies to p53 [76]

DNA in Plasma/Serum

Mutant ras [79] Mutant p53 [90]

Exfoliated Cells p53 Mutations [93,97]

Telomerase activity [84-86]

Loss of heterozygosity [91,92]

Microsatellite alterations [92,93]

mRNA in tumor cells in blood or urine [87-89]

compared to controls. Thus, the relationship between tumor and serum assays requires further validation. In healthy workers with occupational exposure to vinyl chloride, a known liver carcinogen, mutant p53 protein was found more frequently in workers with higher exposure [75]. These results suggest that mutant p53 protein in serum might be an early indicator of risk. Antibodies to p53 protein have also been measured in about 30% of cases with cancer at various sites [reviewed in [76]]. Their presence in high-risk individuals such as exposed workers and smokers suggests that they may also be useful in the early detection of cancer. However, they are not site specific and may present problems in terms of accurate diagnosis.

A strong dose-response relationship between vinyl-chloride exposure and mutant ras p21 protein has also been observed [reviewed in [77]]. In a prospective study, p21 k-ras and p52 SIS were more frequently found in serum of subjects who went on to develop cancer, compared to healthy controls [78]. Plasma DNA has also been analyzed for mutations in k-ras [79]. In patients undergoing colonoscopy, 39% with mutations in plasma had neoplasms with k-ras mutation compared with 3% of patients without mutations. A major concern of this research is the meaning of positive assays in apparently healthy individuals. Studies have also been performed on c-erbB-2 (HER2/neu) [Reviewed in [80]].

Other Markers of Early Stage Disease

Telomerase contributes to the maintenance of telomere stability; its expression can be detected in 85 to 90% of primary cancer tissues [81]. Although it is not clear at what stage of cancer development telomerase is activated, measurement of telomerase activity may be useful for early diagnosis of cancer. Activity has been measured by the telomeric repeat amplification protocol (TRAP); an RNA component can be measured by in situ hybridization [82,83]. A study of exfoliated cells in urine from patients with bladder cancer found that measurement of telomerase activity was more sensitive in detecting the presence of cancer than standard cytologic examination [84]. However, a study in oral mucosa found activity in normal oral squamous epithelium, leukoplakia and carcinomas [85]. Similarly, a study of benign breast lesions (fibroadenoma and dysplasia) obtained by fine-needle aspiration found that 16% of samples were positive compared to 39% of carcinomas using strict criteria [86]. Further studies are necessary to determine the utility of this assay in detecting cancer precursors.

The detection of circulating tumor cells in blood, urine, and pleural effusions has been achieved using reverse transcriptase PCR (RT-PCR) methods to detect, for example, specific mRNAs including a-fetoprotein, a CD44 variant, and two tissue kallikrein family genes [87-89]. Loss of heterozygosity and microsatellite instability have also been used to identify tumor cells or tumor DNA in blood, exfoliated oral cells, and sputum [for example [90-92]]. The recent development of microar-ray technology should provide new ways to investigate early stage disease in apparently healthy individuals. Determination of the complex profiles of gene expression in precursor lesions and in tumors may also provide new insights into individual susceptibility and causative factors [for example [93-96]]. Advances in analyzing complex profiles of cellular proteins ("proteomics") should also provide similar insights.

Conclusion

Advances in basic research on the cellular and molecular mechanisms of carcinogenesis have contributed to our understanding of this complex multistage disease. This research has also led to the development of methods to monitor humans for early stages of disease. Studies on DNA and protein adducts have demonstrated associations of higher levels of adducts with disease status. However, these results have not been applicable to the individual; higher DNA adducts are not proof of higher risk. More-recent studies, identifying tumor cells or mutated DNA in biospecimens, hold promise for the early identification of subjects with preclinical disease. Considerably more research will be necessary however, before these assays can be used for routine medical care.

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