Showing papers on "Carcinogenesis published in 1995"

Expression of Cyclooxygenase-1 and -2 in Human Colorectal Cancer

Abstract: Several studies indicate that nonsteroidal anti-inflammatory drugs including indomethacin, aspirin, sulindac, and piroxicam reduce the risk of colon cancer. Furthermore, nonsteroidal anti-inflammatory drugs that inhibit the cyclooxygenase (COX) enzyme were shown to inhibit the development of colon cancer in animal models of carcinogenesis. Non-steroidal anti-inflammatory drugs inhibit the enzymatic activity of both the constitutive (COX-1) and inducible (COX-2) isoforms of COX enzyme. We have investigated the expression of COX-1 and COX-2 polypeptides in human colon cancer tissues using immunohistochemistry. Enhanced COX-2 expression was observed in colon cancer tissues from 15 subjects with clinically diagnosed colorectal cancer. Marked COX-2 expression was observed in cancer cells, inflammatory cells, vascular endothelium, and fibroblasts of the lesional tissues compared with the nonlesional and normal colon tissues. The extent and intensity of the immunoreactive COX-2 in cancer cells was much greater than that of the other cell types. In contrast, the expression of COX-1 polypeptide was weak in both normal and cancerous specimens. These data suggest that the enhanced expression of the COX-2 gene in colon cancer tissues may contribute to the enhanced synthesis of prostaglandin E2 by the colon cancer tissues. Enhanced expression of COX-2 may play a role in the pathogenesis of colon cancer. Furthermore, selective inhibition of COX-2 may prove to be more efficacious in the retardation of colon cancer development.

Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16

Abstract: D-type cyclins, in association with the cyclin-dependent kinases Cdk4 or Cdk6, promote progression through the G1 phase of the cell cycle by phosphorylating the retinoblastoma protein (RB). The activities of Cdk4 and Cdk6 are constrained by inhibitors such as p16, the product of the CDKN2 gene on human chromosome 9p21 (refs 12-14). The frequent deletion or mutation of CDKN2 in tumour cells suggests that p16 acts as a tumour suppressor. We show that wild-type p16 arrests normal diploid cells in late G1, whereas a tumour-associated mutant of p16 does not. Significantly, the ability of p16 to induce cell-cycle arrest is lost in cells lacking functional RB, including primary fibroblasts from Rb-/- mouse embryos. Thus, loss of p16, overexpression of D-cyclins and loss of RB have similar effects on G1 progression, and may represent a common pathway to tumorigenesis.

Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas.

Abstract: The AKT2 gene is one of the human homologues of v-akt, the transduced oncogene of the AKT8 virus, which induces lymphomas in mice. In previous studies, AKT2, which codes for a serine-threonine protein kinase, was shown to be amplified and overexpressed in some human ovarian carcinoma cell lines and amplified in primary tumors of the ovary. To confirm and extend these findings, we conducted a large-scale, multicenter study of AKT2 alterations in ovarian and breast cancer. Southern-blot analysis demonstrated AKT2 amplification in 16 of 132 (12.1%) ovarian carcinomas and in 3 of 106 (2.8%) breast carcinomas. No AKT2 alteration was detected in 24 benign or borderline tumors. Northern-blot analysis revealed overexpression of AKT2 in 3 of 25 fresh ovarian carcinomas which were negative for AKT2 amplification. The difference in the incidence of AKT2 alterations in ovarian and breast cancer suggests a specific role for this gene in ovarian oncogenesis. No significant association was found between AKT2 amplification and amplification of the proto-oncogenes MYC and ERBB2, suggesting that amplification of AKT2 defines an independent subset of breast and ovarian cancers. Ovarian cancer patients with AKT2 alterations appear to have a poor prognosis. Amplification of AKT2 was especially frequent in undifferentiated tumors (4 of 8, p = 0.019), suggesting that AKT2 alterations may be associated with tumor aggressiveness.

Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B

Abstract: Multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma are dominantly inherited cancer syndromes. All three syndromes are associated with mutations in RET, which encodes a receptor-like tyrosine kinase. The altered RET alleles were shown to be transforming genes in NIH 3T3 cells as a consequence of constitutive activation of the RET kinase. The MEN2A mutation resulted in RET dimerization at steady state, whereas the MEN2B mutation altered RET catalytic properties both quantitatively and qualitatively. Oncogenic conversion of RET in these neoplastic syndromes establishes germline transmission of dominant transforming genes in human cancer.

Induction of apoptosis by the Bcl-2 homologue Bak

Abstract: CELLS are eliminated in a variety of physiological settings by apoptosis, a genetically encoded process of cellular suicide1,2. Apoptosis comprises an intrinsic cellular defence against tumorigenesis, which, when suppressed, may contribute to the development of malignancies3. The bcl-2oncogene, which is activated in follicular lymphomas, functions as a potent suppressor of apoptosis under diverse conditions4. Here we describe the complementary DNA cloning and functional analysis of a new Bcl-2 homologue, Bak, which promotes cell death and counteracts the protection from apoptosis provided by Bcl-2. Moreover, enforced expression of Bak induces rapid and extensive apoptosis of serum-deprived fibroblasts. This raises the possibility that Bak is directly involved in activating the cell death machinery.

Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis.

Abstract: To investigate the relationship between angiogenesis and hepatic tumorigenesis, we examined the expression of vascular endothelial growth factor (VEGF) in 8 human colon carcinoma cell lines and in 30 human colorectal cancer liver metastases. Abundant message for VEGF was found in all tumors, localized to the malignant cells within each neoplasm. Two receptors for VEGF, KDR and flt1, were also demonstrated in most of the tumors examined. KDR and flt1 mRNA were limited to tumor endothelial cells and were more strongly expressed in the hepatic metastases than in the sinusoidal endothelium of the surrounding liver parenchyma. VEGF monoclonal antibody administration in tumor-bearing athymic mice led to a dose- and time-dependent inhibition of growth of subcutaneous xenografts and to a marked reduction in the number and size of experimental liver metastases. In hepatic metastases of VEGF antibody-treated mice, neither blood vessels nor expression of the mouse KDR homologue flk-1 could be demonstrated. These data indicate that VEGF is a commonly expressed angiogenic factor in human colorectal cancer metastases, that VEGF receptors are up-regulated as a concomitant of hepatic tumorigenesis, and that modulation of VEGF gene expression or activity may represent a potentially effective antineoplastic therapy in colorectal cancer.

Calcium-mediated cell injury and cell death.

Abstract: The effect of intracellular ion deregulation, particularly of [Ca2+], on the events following acute cell injury and the progression of change from initiation (reversible) to maintenance (reversible-irreversible) phases and finally to cell death has been the major thrust of experimentation in our laboratory for over 20 years. Cell death, which plays an important role in both normal and pathological phenomena, has been classified into two principal types, accidental and programmed. Recent exploration of programmed cell death (or apoptosis) has revealed extensive data showing it is an important mechanism for the normal maintenance and also differentiation of a variety of cell types and organs. From the results from our laboratory and those of others, we continue to expand and refine our working hypothesis: deregulation of [Ca2+] results in a number of phenomena from activation of signaling mechanisms and alterations in cellular structure to alterations in gene expression, all of which contribute to or play a critical role in cellular toxicity, including carcinogenesis and cell death. Therefore, although much more experimentation is needed to clarify some of these phenomena, the implications of such data for understanding the mechanisms and processes involved in carcinogenesis and the chemotherapeutic killing of cancer cells are extremely exciting. These relationships between [Ca2+], cell injury, and cell death are briefly reviewed here within the framework of our hypothesis.

Decreased expression of BRCA1 accelerates growth and is often present during sporadic breast cancer progression

Abstract: We have characterized expression of the familial breast and ovarian cancer gene, BRCA1, in cases of non-hereditary (sporadic) breast cancer and analyzed the effect of antisense inhibition of BRCA1 on the proliferative rate of mammary epithelial cells. BRCA1 mRNA levels are markedly decreased during the transition from carcinoma in situ to invasive cancer. Experimental inhibition of BRCA1 expression with antisense oligonucleotides produced accelerated growth of normal and malignant mammary cells, but had no effect on non-mammary epithelial cells. These studies suggest that BRCA1 may normally serve as a negative regulator of mammary epithelial cell growth whose function is compromised in breast cancer either by direct mutation or alterations in gene expression.

Interaction between the retinoblastoma protein and the oncoprotein MDM2

Abstract: Inactivation of tumour-suppressor genes leads to deregulated cell proliferation and is a key factor in human tumorigenesis. Both p53 and retinoblastoma genes are frequently mutated in human cancers, and the simultaneous inactivation of RB and p53 is frequently observed in a variety of naturally occurring human tumours. Furthermore, three distinct DNA tumour virus groups--papovaviruses, adenoviruses and human papillomaviruses--transform cells by targeting and inactivating certain functions of both the p53 and retinoblastoma proteins. The cellular oncoprotein, Mdm2, binds to and downmodulates p53 function; its human homologue, MDM2, is amplified in certain human tumours, including sarcomas and gliomas. Overproduction of Mdm2 is both tumorigenic and capable of immortalizing primary rat embryo fibroblasts. Here we show that MDM2 interacts physically and functionally with pRB and, as with p53, inhibits pRB growth regulatory function. Therefore, both pRB and p53 can be subjected to negative regulation by the product of a single cellular protooncogene.

Topological Control of p21WAF1/CIP1 Expression in Normal and Neoplastic Tissues

Abstract: The p53-regulated gene product p21WAF1/CIP1 is the prototype of a family of small proteins that negatively regulate the cell cycle. To learn more about p21WAF1/CIP1 regulation in vivo, monoclonal antibodies were developed for immunohistochemistry. These revealed that p21WAF1/CIP1 expression followed radiation-induced DNA damage in human skin in a pattern consistent with its regulation by p53. A detailed comparison of the human, rat, and mouse p21WAF1/CIP1 promoter sequences revealed that this induction was probably mediated by conserved p53-binding sites upstream of the transcription start site. In unirradiated tissues, p21WAF1/CIP1 expression was apparently independent of p53 and was observed in a variety of cell types. Moreover, there was a striking compartmentalization of p21WAF1/CIP1 expression throughout the gastrointestinal tract that correlated with proliferation rather than differentiation. As epithelial cells migrated up the crypts, the Ki67-expressing proliferating compartment near the crypt base ended abruptly, with the coincident appearance of a nonproliferating compartment expressing p21WAF1/CIP1. In colonic neoplasms, this distinct compartmentalization was largely abrogated. Cell cycle inhibitors are thus subject to precise topological control, and escape from this regulation may be a critical feature of neoplastic transformation.

bcl-2 and p53 Oncoprotein Expression during Colorectal Tumorigenesis

Abstract: Apoptosis or programmed cell death represents a mechanism by which cells possessing DNA damage can be deleted. The bcl-2 proto-oncogene is a known inhibitor of apoptosis that may allow the accumulation and propagation of cells containing genetic alterations. To determine if and when the bcl-2 gene is activated during colorectal tumorigenesis and its relationship to p53, we analyzed normal mucosa, hyperplastic and dysplastic epithelial polyps, and carcinomas for the expression of these markers using immunohistochemistry. Whereas bcl-2 staining was restricted to basal epithelial cells in normal and hyperplastic mucosa, bcl-2 expression was detected in parabasal and superficial regions in dysplastic polyps and carcinomas. An inverse correlation was found between bcl-2 and p53 expression in adenomas, suggesting that these markers may regulate a common cell death pathway. Furthermore, carcinomas with a high percentage of bcl-2-positive cells were significantly more likely to have low rates of spontaneous apoptosis, as determined histologically, than those cancers with low or absent bcl-2 expression. Abnormal activation of the bcl-2 gene appears to be an early event in colorectal tumorigenesis that can inhibit apoptosis in vivo and may facilitate tumor progression.

Inhibition of apoptosis during development of colorectal cancer.

Abstract: Colorectal tumorigenesis proceeds through an accumulation of specific genetic alterations. Studies of the mechanism by which these genetic changes effect malignant transformation have focused on the deregulation of cell proliferation. However, colorectal epithelial homeostasis is dependent not only on the rate of cell production but also on apoptosis, a genetically programmed process of autonomous cell death. We investigated whether colorectal tumorigenesis involved an altered susceptibility to apoptosis by examining colorectal epithelium from normal mucosa, adenomas from familial adenomatous polyposis, sporadic adenomas, and carcinomas. The transformation of colorectal epithelium to carcinomas was associated with a progressive inhibition of apoptosis. The inhibition of apoptosis in colorectal cancers may contribute to tumor growth, promote neoplastic progression, and confer resistance to cytotoxic anticancer agents.

Tumour-derived p16 alleles encoding proteins defective in cell-cycle inhibition.

Abstract: The cyclin-dependent kinase inhibitor p16 is a candidate tumour-suppressor protein that maps to a genomic locus strongly associated with familial melanoma and other tumour types. Screening of primary tumours and linkage analysis of familial melanoma pedigrees have identified many potential mutations in p16, but the functional significance of these sequence variants has remained unclear. We report here that p16 can act as a potent and specific inhibitor of progression through the G1 phase of the cell cycle, and we demonstrate that several tumour-derived alleles of p16 encode functionally compromised proteins. The ability of p16 to arrest cell-cycle progression generally correlates with inhibition of cyclin D1/Cdk4 kinase activity in vitro, with two exceptions among the alleles tested. In vivo, the presence of functional retinoblastoma protein appears to be necessary but may not be sufficient to confer full sensitivity to p16-mediated growth arrest. Our results provide support for the notion that p16 is an important cell-cycle regulator whose inactivation contributes to the outgrowth of human tumours.

Expression of the beta 6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling.

Abstract: The alpha v beta 6 integrin was identified in cultured epithelial cells and functions as a fibronectin receptor. We have now used monoclonal antibodies to determine in vivo expression patterns of the beta 6 subunit in normal and pathological human or primate tissues, and during experimental wound healing or induced lung injury. The results indicate that beta 6 expression is restricted to epithelia and is up-regulated in parallel with morphogenetic events, tumorigenesis, and epithelial repair. During development of the kidney, lung, and skin, we found that beta 6 is expressed by specific types of epithelial cells, whereas it is mostly undetectable in normal adult kidney, lung and skin. In contrast, we detected high-level expression in several types of carcinoma. For example, beta 6 is almost invariably neo-expressed in squamous cell carcinomas derived from the oral mucosa, often focally localized at the infiltrating edges of tumor islands. Expression of beta 6 is also upregulated in migrating keratinocytes at the wound edge during experimental epidermal wound healing. Similarly, beta 6 expression is induced in type II alveolar epithelial cells during lung injury caused by injection of live bacteria. We also observed beta 6 expression in adult lungs and kidneys at focal sites of subclinical inflammation, as well as in a variety of clinical specimens from patients with chronic or acute inflammation of the lungs or kidneys. From these findings and earlier results, we hypothesize that alpha v beta 6 affects cell spreading, migration and growth during reorganization of epithelia in development, tissue repair, and neoplasia.

Telomerase activity in gastric cancer.

Abstract: Although many genetic alterations have been reported in gastric cancer, it is not known whether all gastric tumors are capable of indefinite proliferative potential, e.g., immortality. The expression of telomerase and stabilization of telomeres are concomitant with the attainment of immortality in tumor cells; thus, the measurement of telomerase activity in clinically obtained tumor samples may provide important information useful both as a diagnostic marker to detect immortal cancer cells in clinical materials and as a prognostic indicator of patient outcome. Telomerase activity was analyzed in 66 primary gastric cancers with the use of a PCR-based assay. The majority of tumors (85%) displayed telomerase activity, but telomerase was undetectable in 10 tumors (15%), 8 of which were early stage tumors. Most of the tumors with telomerase activity were large and of advanced stages, including metastases. Survival rate of patients of tumors with detectable telomerase activity was significantly shorter than that of those without telomerase activity. Alterations of telomere length (reduced/elongated terminal restriction fragments) were detected in 14 of 66 (21%) gastric cancers, and all 14 had telomerase activity. Cellular DNA contents revealed that all 22 aneuploid tumors had detectable telomerase activity. The present results indicate that telomerase activation may be required as a critical step in the multigenetic process of tumorigenesis, and that telomerase is frequently but not always activated as a late event in gastric cancer progression.

Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors

Abstract: Neuroblastoma (NB), a tumor arising from the sympathetic nervous system, is one of the most common malignancies in childhood. Several recent reports on the p53 genotype found virtually exclusive wild-type status in primary tumors, and it was postulated that p53 plays no role in the development of NB. Here, however, we report that the vast majority of undifferentiated NBs exhibit abnormal cytoplasmic sequestration of wild-type p53. This inability of p53 to translocate to the nucleus presumably prevents the protein from functioning as a suppressor. Thirty of 31 cases (96%) of undifferentiated NB showed elevated levels of wild-type p53 in the cytoplasm of all tumor cells concomittant with a lack of nuclear staining. p53 immunoprecipitation from tumor tissues showed a 4.5- to 8-fold increase over normal protein levels. All of 10 tumors analyzed harbored wild-type p53 by direct sequencing of full-length cDNA and Southern blot. In addition, no MDM-2 gene amplification was seen in all 11 tumors analyzed. In contrast, no p53 abnormality was detected in 14 differentiated ganglioneuroblastomas and 1 benign ganglioneuroma. We conclude that loss of p53 function seems to play a major role in the tumorigenesis of undifferentiated NB. This tumor might abrogate the transactivating function of p53 by inhibiting its access to the nucleus, rather than by gene mutation. Importantly, our results suggest that (i) this could be a general mechanism for p53 inactivation not limited to breast cancer (where we first described it) and that (ii) it is found in a tumor previously not thought to be affected by p53 alteration.

Contributions of tumor and stromal matrix metalloproteinases to tumor progression, invasion and metastasis.

Abstract: The malignant progression of tumors is driven by the expression of oncogenes and loss of expression of tumor suppressor genes; factors that are intrinsic to cancer cells. The phenotypic changes brought about by the gain or loss of expression of oncogenes and tumor suppressor genes lead to the acquisition of malignant traits, namely, the ability to invade into and grow in ectopic tissue environments. Recently, however, focus in cancer research has widened from the cancer cell to include the surrounding tumor stroma as an integral player in the process of tumor progression. One of the areas in cancer research contributing to this enhanced appreciation of stromal involvement in tumor progression and metastasis is that of matrix metalloproteinases (MMPs). This review provides an overview of the characteristics of MMPs and discusses their role in the progression and metastasis of tumors. Initially, attention will focus on the regulation of MMPs in tumor cells but will switch to discourse on stromal expression of MMPs in tumors and speculation on the functional consequences of stromal expression of MMPs.

Regulation of Lymphocyte Survival by the BCL-2 Gene Family

Abstract: The control of cell survival is of central importance in tissues with high cell turnover such as the lymphoid system, and its disruption may be a critical step in tumorigenesis. Genes homologous to bcl-2, the oncogene implicated in human follicular lymphoma, play a key role in regulating physiologic cell death (apoptosis). Bcl-2 and its relatives bcl-x and bax encode intracellular membrane-bound proteins that share homology in three domains with a wider family of viral and cellular proteins. The Bcl-2 and Bcl-x proteins enhance the survival of lymphocytes and other cell types but do not promote their proliferation. High levels of Bax or of a smaller Bcl-x variant antagonize the survival function of Bcl-2. The mechanism by which Bcl-2 promotes cell survival remains unknown, but it appears to require association with Bax. Bcl-2 may combat the action of cysteine proteases thought to trigger apoptosis. Bcl-2 is not essential for embryogenesis or lymphoid development. However, upregulation of Bcl-2 appears to be the normal mechanism for positive selection of developing lymphocytes, and its continued expression is critical for survival of mature peripheral B and T cells. Constitutive expression of Bcl-2 does not abrogate deletion of self-reactive lymphocytes, nor disturb T lymphoid homeostasis; however, it substantially increases the pool of mature noncycling B cells. The risk of B lymphoid tumors is also enhanced, probably because Bcl-2 can countermand the apoptotic action of other oncoproteins such as Myc. Expression in tumors of bcl-2 and other cell survival genes may constitute a major barrier to the success of genotoxic cancer therapy.

Growth factor modulation of p53-mediated growth arrest versus apoptosis.

Abstract: Irradiation of mammalian cells can cause cell cycle perturbations and apoptotic cell death. We have investigated the modulation of these physiologic end points by growth factor stimulation: irradiation of a murine hematopoietic cell line in the presence of interlekin-3 (IL-3) induces G1 arrest, and irradiation in the absence of IL-3 results in rapid apoptotic cell death. Both of these end points are dependent on p53. Transient removal of IL-3 at the time of irradiation results in decreased clonogenic survival of irradiated cells. The removal of IL-3 results in a failure of the irradiated cells to arrest at the G1 checkpoint, despite induction of p53 and p21WAF1/CIP1, and then the cells enter S-phase where they undergo apoptosis. There are no cytokine-related changes in Bcl-2, Bax, or Bcl-x protein levels that could account for the modulation of G1 arrest versus apoptosis by growth factors. In contrast, rapid p53-independent alterations of basal levels of gadd45 and p21WAF1/CIP1 expression are linked to IL-3 withdrawal, suggesting a potential mechanism for this modulation. Constitutive activation of cytokine-like pathways with induced expression of v-Src or activated c-Raf inhibits the radiation-induced apoptosis and the alterations in p21WAF1/CIP1 and gadd45 expression. These observations suggest additional molecular mechanisms that can contribute to the development of radioresistance and resistance to apoptosis during tumorigenesis and provide an explanation for the observed lack of p53 mutations in some tumor types. In addition, these data suggest that oncogenic changes occurring during multistep tumorigenesis could be classified as those that either enhance or decrease apoptosis tendencies.

Somatic mutations in the BRCA1 gene in sporadic ovarian tumours.

Abstract: The BRCA1 gene on chromosome 17q21 is responsible for an autosomal dominant syndrome of increased susceptibility to breast and ovarian cancer but no somatic mutations in tumours have yet been described. To study the potential role of BRCA1 in sporadic carcinogenesis, we analysed the genomic DNA of tumour and normal fractions of 47 ovarian cancers for mutations in BRCA1 using the single-strand conformation polymorphism technique. We now describe somatic mutations in the DNA of four tumours which also had loss of heterozygosity (LOH) at a BRCA1 intragenic marker. Our data support a tumour suppressor mechanism for BRCA1; somatic mutations and LOH may result in inactivation of BRCA1 in at least a small number of ovarian cancers.

Involvement of the p53 tumor suppressor in repair of u.v.-type DNA damage

Abstract: The tumor suppressor p53 plays a central role in the cellular responses to genotoxic stress. Besides its well known role in activation of the G1 checkpoint after exposure to agents like ionizing radiation and its role in apoptosis, the possibility exists that p53 may have additional roles, such as in DNA repair. For example, p53, is known to bind to single strand DNA such as would occur during repair events, and the proteins encoded by two p53-regulated genes have previously been found to bind to at least one protein involved in DNA damage processing including nucleotide excision repair (NER). NER is an important and versatile DNA repair mechanism, which is the major pathway for repair of u.v.-type lesions and damage by a variety of important carcinogens and mutagens. If components of the p53 pathway are involved in NER, then disruption of p53 function by mutations or expression of certain viral proteins could have important implications in carcinogenesis and cancer treatment. In the present study we show that disruption of normal p53 function in human colon carcinoma RKO cells with either the human papillomavirus E6 oncoprotein or a dominant-negative mutant p53 transgene results in reduced repair of u.v.-induced DNA damage. The E6 and mutant p53-containing cell lines demonstrated reduced repair of u.v.-induced DNA lesions in host cell reactivation experiments with reporter plasmids, and reduced repair in in vitro DNA repair assays. With this in vitro assay, extracts from the E6- and mutant p53-containing lines also showed loss of induced repair following cellular u.v.-irradiation. The reduced DNA repair activity of the transfected cell lines also correlated with reduced clonogenic survival following u.v.-irradiation. These results indicate that p53 and/or p53-regulated gene products function in the NER pathway and that this process is inducible by DNA damage.

Telomerase activity associated with acquisition of malignancy in human colorectal cancer.

Abstract: Shortening of telomeres may contribute to the control of the proliferative capacity of normal cells, and telomerase, the enzyme that elongates telomeric DNA, may be essential for unlimited cell proliferation. We have shown previously that telomerase activity is present in human cells immortalized in vitro and in metastatic ovarian carcinoma cells but is undetectable in normal cultured cells or normal tissues. We have determined the temporal pattern of telomerase activity during colorectal carcinogenesis in man. We report that telomerase activity is associated with acquisition of malignancy as it is detectable in colorectal carcinoma but not in adenomatous polyps. Mutations leading to reactivation or upregulation of the enzyme may represent an additional required event in the multistep development of colorectal cancer.

Effect of genistein on in vitro and in vivo models of cancer.

Abstract: In two-thirds of studies on the effect of genistein-containing soy materials in animal models of cancer, the risk of cancer (incidence, latency or tumor number) was significantly reduced. In addition, purified genistein delayed mammary tumor appearance in association with increased cell differentiation in mammary tissue in rats treated with 7, 12-dimethylbenz[a]anthracene when administered neonatally, inhibited phorbol ester-induced H2O2 production in a model of skin cancer, and inhibited aberrant crypt formation in a model of colonic cancer. In in vitro models, genistein inhibited the proliferation of human tumor cell lines in culture with a wide variation in IC50 values (2.6-79 mumol/L, or 1-30 micrograms/mL). In only a few cases was the IC50 below 13.2 mumol/L (5 micrograms/mL), the presumed upper limit for the serum genistein concentration in those on a high soy diet. In future studies, greater emphasis should be placed on the effect of genistein on nontransformed, normal cell lines from the tissues where cancer can occur rather than established tumor cell lines. Similarly, the effect of genistein on the progression and/or promotion of cancer may be more clearly examined using nontransformed cell lines transfected with specific oncogenes thought to be activated during oncogenesis.

M6P/IGF2R gene is mutated in human hepatocellular carcinomas with loss of heterozygosity.

Abstract: The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) functions in the intracellular trafficking of lysosomal enzymes1, the activation of the potent growth inhibitor, transforming growth factor β2, and the degradation of IGF2 (ref. 1), a mitogen often overproduced in tumours3–6. We have recently shown that 70% of human hepatocellular tumours have loss of heterozygosity (LOH) at the M6P/IGF2R locus7 which maps to chromosome 6q26–q27 (ref. 8). Using a coarse screen, we have now identified point mutations in the remaining allele of 25% of human hepatocellular carcinomas (HCCs) with LOH. These mutations give rise to truncated receptor protein and significant amino acid substitutions, and provide evidence that the M6P/IGF2R gene functions as a tumour suppressor in human liver carcinogenesis.

Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens.

Abstract: A transgenic gpt+ Chinese hamster cell line (G12) was found to be susceptible to carcinogenic nickel-induced inactivation of gpt expression without mutagenesis or deletion of the transgene. Many nickel-induced 6-thioguanine-resistant variants spontaneously reverted to actively express gpt, as indicated by both reversion assays and direct enzyme measurements. Since reversion was enhanced in many of the nickel-induced variant cell lines following 24-h treatment with the demethylating agent 5-azacytidine, the involvement of DNA methylation in silencing gpt expression was suspected. This was confirmed by demonstrations of increased DNA methylation, as well as by evidence indicating condensed chromatin and heterochromatinization of the gpt integration site in 6-thioguanine-resistant cells. Upon reversion to active gpt expression, DNA methylation and condensation are lost. We propose that DNA condensation and methylation result in heterochromatinization of the gpt sequence with subsequent inheritance of the now silenced gene. This mechanism is supported by direct evidence showing that acute nickel treatment of cultured cells, and of isolated nuclei in vitro, can indeed facilitate gpt sequence-specific chromatin condensation. Epigenetic mechanisms have been implicated in the actions of some nonmutagenic carcinogens, and DNA methylation changes are now known to be important in carcinogenesis. This paper further supports the emerging theory that nickel is a human carcinogen that can alter gene expression by enhanced DNA methylation and compaction, rather than by mutagenic mechanisms.

Functional inactivation but not structural mutation of p53 causes liver cancer

Abstract: Structural mutations in the p53 gene are seen in virtually every form of human cancer. To determine whether such mutations are important for initiating tumorigenesis, we have been studying hepatocellular carcinoma, in which most cases are associated with chronic hepatitis B virus infections. Using a transgenic mouse model where expression of a single HBV gene product, the HBx protein, induces progressive changes in the liver, we show that tumour development correlates precisely with p53 binding to HBx in the cytoplasm and complete blockage of p53 entry into the nucleus. Analysis of tumour cell DNA shows no evidence for p53 mutation, except in advanced tumours where a small proportion of cells may have acquired specific base substitutions. Our results suggest that genetic changes in p53 are late events which may contribute to tumour progression.