Mammalian cells resistant to tumor suppressor genes.

Expression of p53 causes growth arrest or apoptosis in many normal and neoplastic cell types, but the relationship between these two effects has remained obscure. To begin to dissect the underlying mechanisms at a genetic level, we have generated mutant cells resistant to the action of wild-type p53. Rat embryo fibroblasts transformed with ras and a temperature-sensitive p53 (tsp53(135val)) gene were chemically mutagenized and selected for growth at a temperature at which p53 adopts a wild-type conformation (31.5 degrees C). Clones that grew exponentially at 31.5 degrees C were selected. Cell fusion experiments demonstrated that the mutations conferring resistance to p53-mediated growth arrest were dominant. The mutagenized clones were resistant not only to p53-mediated growth arrest, but also to the apoptosis induced by E1A in conjunction with p53, and partially resistant to the retinoblastoma tumor suppressor, pRB. The results suggest that a single downstream pathway can control the induction of growth arrest and apoptosis, and that both p53 and RB function through this pathway.

Modulation of the transcriptional activity of thyroid hormone receptors by the tumor suppressor p53.

Thyroid hormone nuclear receptors (TRs) are ligand-dependent transcriptional factors that regulate growth, differentiation, and development. The molecular mechanisms by which TRs mediate these effects are unclear. One prevailing hypothesis suggests that TRs may cooperate with other transcriptional factors to mediate their biological effects. In this study, we tested this hypothesis by examining whether the activity of TRs is modulated by the tumor suppressor p53. p53 is a nuclear protein that regulates gene expression via sequence-specific DNA binding and/or direct protein-protein interaction. We found that the human TR subtype beta 1 (h-TR beta 1) physically interacted with p53 via its DNA binding domain. As a result of this physical interaction, binding of h-TR beta 1 to its hormone response elements either as homodimer or as a heterodimer with the retinoic X receptor was inhibited by p53 in a concentration-dependent manner. In transfected cells, wild-type p53 repressed the hormone-dependent transcriptional activation of h-TR beta 1. In contrast, mutant p53 either had no effect or activated the transcriptional activity of h-TR beta 1 depending on the type of hormone response elements. These results indicate the gene regulating activity of TRs was modulated by p53, suggesting that the cross talk between these two transcriptional factors may play an important role in the biology of normal and cancer cells.

Dietary lipids and calorie restriction affect mammary tumor incidence and gene expression in mouse mammary tumor virus/v-Ha-ras transgenic mice.

We have studied the effects of food restriction (FR) and substitution of fish oil (FO; omega 3) for corn oil (CO; omega 6) on breast tumor incidence and survival in mouse mammary tumor virus/v-Ha-ras transgenic (Onco) mice. The diets were as follows: group 1, 5% (wt/wt) CO fed ad libitum (AL); group 2, 5% CO, restricted calories (40% fewer calories than AL; FR); group 3, 20% CO fed AL; and group 4, 20% FO fed AL. After 3 years, 40% of FR Onco (group 2) mice were alive, whereas there were no survivors in the other three groups. Similarly, tumor incidence was reduced to 27% (5 out of 18) in FR animals (group 2), whereas it was 83% (11 out of 13) in group 1 mice, 89% (16 out of 18) in group 3 mice, and 71% (10 out of 14) in group 4 mice. These protective effects of FR on survival and tumor incidence were paralleled by higher expression of the tumor suppressor gene p53 (wild type) and free-radical scavenging enzymes (catalase and superoxide dismutase) in breast tumors. Immunoblotting showed less ras gene product, p21, and increased p53 levels in the tumors of FR mice. In addition, FR decreased RNA levels of c-erbB-2, interleukin 6, and the transgene v-Ha-ras in tumors. In contrast, analysis of hepatic mRNA from tumor-bearing FR mice revealed higher expression of catalase, glutathione peroxidase, and superoxide dismutase. Survival and tumor incidence were not influenced significantly by dietary supplementation with FO in place of CO. Taken together, our studies suggest that moderate restriction of energy intake significantly inhibited the development of mammary tumors and altered expression of cytokines, oncogenes, and free-radical scavenging enzymes.

HLS5, a novel RBCC (ring finger, B box, coiled-coil) family member isolated from a hemopoietic lineage switch, is a candidate tumor suppressor

Hemopoietic cells, apparently committed to one lineage, can be reprogrammed to display the phenotype of another lineage. The J2E erythroleukemic cell line has on rare occasions developed the features of monocytic cells. Subtractive hybridization was used in an attempt to identify genes that were up-regulated during this erythroid to myeloid transition. We report here on the isolation of hemopoietic lineage switch 5 (Hls5), a gene expressed by the monocytoid variant cells, but not the parental J2E cells. Hls5 is a novel member of the RBCC (Ring finger, B box, coiled-coil) family of genes, which includes Pml, Herf1, Tif-1alpha, and Rfp. Hls5 was expressed in a wide range of adult tissues; however, at different stages during embryogenesis, Hls5 was detected in the branchial arches, spinal cord, dorsal root ganglia, limb buds, and brain. The protein was present in cytoplasmic granules and punctate nuclear bodies. Isolation of the human cDNA and genomic DNA revealed that the gene was located on chromosome 8p21, a region implicated in numerous leukemias and solid tumors. Enforced expression of Hls5 in HeLa cells inhibited cell growth, clonogenicity, and tumorigenicity. It is conceivable that HLS5 is one of the tumor suppressor genes thought to reside at the 8p21 locus.

An inflammatory role for the mammalian carboxypeptidase inhibitor latexin: Relationship to cystatins and the tumor suppressor TIG1

Latexin, the only known mammalian carboxypeptidase inhibitor, has no detectable sequence similarity with plant and parasite inhibitors, but it is related to a human putative tumor suppressor protein, TIG1. Latexin is expressed in the developing brain, and we find that it plays a role in inflammation, as it is expressed at high levels and is inducible in macrophages in concert with other protease inhibitors and potential protease targets. The crystal structure of mouse latexin, solved at 1.83 Angstrom resolution, shows no structural relationship with other carboxypeptidase inhibitors. Furthermore, despite a lack of detectable sequence duplication, the structure incorporates two topologically analogous domains related by pseudo two-fold symmetry. Surprisingly, these domains share a cystatin fold architecture found in proteins that inhibit cysteine proteases, suggesting an evolutionary and possibly functional relationship. The structure of the tumor suppressor protein TIG1 was modeled, revealing its putative membrane binding surface.

The PCNA-associated factor KIAA0101/p15(PAF) binds the potential tumor suppressor product p33ING1b

The KIAA0101/p15(PAF)/OEATC-1 protein was initially isolated in a yeast two-hybrid screen for proliferating cell nuclear antigen (PCNA) binding partners, and was shown to bind PCNA competitively with the cell cycle regulator p21(WAF). PCNA is involved in DNA replication and damage repair. Using polyclonal antisera raised against a p15(PAF) fusion protein, we have shown that in a range of mammalian tumor and non-tumor cell lines the endogenous p15(PAF) protein localises to the nucleus and the mitochondria. Under normal conditions no co-localisation with PCNA could be detected, however following exposure to UV it was possible to co-immunoprecipitate p15(PAF) and PCNA from a number of cell lines, suggesting a UV-enhanced association of the two proteins. Overexpression of p15(PAF) in mammalian cells was also found to protect cells from UV-induced cell death. Based on similarities between the behaviour of p15(PAF) and the potential tumor suppressor product p33ING1b, we have further shown that these two proteins interact in the same complex in cell cultures. This suggests that p15(PAF) forms part of a larger protein complex potentially involved in the regulation of DNA repair, apoptosis and cell cycle progression. (c) 2005 Elsevier Inc. All rights reserved.

Polymorphisms and DNA methylation of gene TP53 associated with extra-axial brain tumors

The p53 tumor suppressor gene is the most frequently mutated gene in human cancer; this gene is mutated in up to 50% of human tumors. It has a critical role in the cell cycle, apoptosis and cell senescence, and it participates in many crucial physiological and pathological processes. Polymorphisms of p53 have been suggested to be associated with genetically determined susceptibility in various types of cancer. Another process involved with the development and progression of tumors is DNA hypermethylation. Aberrant methylation of the promoter is an alternative epigenetic change in genetic mechanisms, leading to tumor suppressor gene inactivation. In the present study, we examined the TP53 Arg72Pro and Pro47Ser polymorphisms using PCR-RFLP and the pattern of methylation of the p53 gene by methylation-specific PCR in 90 extra-axial brain tumor samples. Patients who had the allele Pro of the TP53 Arg72Pro polymorphism had an increased risk of tumor development ( odds ratio, OR = 3.23; confidence interval at 95%, 95% CI = 1.71-6.08; P = 0.003), as did the allele Ser of TP53 Pro47Ser polymorphism (OR = 1.28; 95% CI = 0.03-2.10; P = 0.01). Comparison of overall survival of patients did not show significant differences. In the analysis of DNA methylation, we observed that 37.5% of meningiomas, 30% of schwannomas and 52.6% of metastases were hypermethylated, suggesting that methylation is important for tumor progression. We suggest that TP53 Pro47Ser and Arg72Pro polymorphisms and DNA hypermethylation are involved in susceptibility for developing extra-axial brain tumors.

Common chromosomal fragile site FRA16D sequence: identification of the FOR gene spanning FRA16D and homozygous deletions and translocation breakpoints in cancer cells

Fluorescence in situ hybridization of a tile path of DNA subclones has previously enabled the cytogenetic definition of the minimal DNA sequence which spans the FRA16D common chromosomal fragile site, located at 16q23.2. Homozygous deletion of the FRA16D locus has been reported in adenocarcinomas of stomach, colon, lung and ovary. We have sequenced the 270 kb containing the FRA16D fragile site and the minimal homozygously deleted region in tumour cells. This sequence enabled localization of some of the tumour cell breakpoints to regions which contain AT-rich secondary structures similar to those associated with the FRA10B and FRA16B rare fragile sites. The FRA16D DNA sequence also led to the identification of an alternatively spliced gene, named FOR (fragile site FRA16D oxidoreductase), exons of which span both the fragile site and the minimal region of homozygous deletion. In addition, the complete DNA sequence of the FRA16D-containing FOR intron reveals no evidence of additional authentic transcripts. Alternatively spliced FOR transcripts (FOR I, FOR II and FOR III) encode proteins which share N-terminal WW domains and differ at their C-terminus, with FOR III having a truncated oxidoreductase domain. FRA16D-associated deletions selectively affect the FOR gene transcripts. Three out of five previously mapped translocation breakpoints in multiple myeloma are also located within the FOR gene. FOR is therefore the principle genetic target for DNA instability at 16q23.2 and perturbation of FOR function is likely to contribute to the biological consequences of DNA instability at FRA16D in cancer cells.

Gene replacement with the human BRCA1 locus: tissue specific expression and rescue of embryonic lethality in mice

We have generated transgenic mice that harbor a 140 kb genomic fragment of the human BRCA1 locus (TgN.BRCA1(GEN)). We find that the transgene directs appropriate expression of human BRCA1 transcripts in multiple mouse tissues, and that human BRCA1 protein is expressed and stabilized following exposure to DIVA damage, Such mice are completely normal, with no overt signs of BRCA1 toxicity commonly observed when BRCA1 is expressed from heterologous promoters. Most importantly, however, the transgene rescues the otherwise lethal phenotype associated with the targeted hypomorphic allele (Brca1(Delta exIISA)). Brca1(-/-); TgN.BRCA1(GEN) bigenic animals develop normally and can be maintained as a distinct line. These results show that a 140 kb fragment of chromosome 17 contains all elements necessary for the correct expression, localization, and function of the BRCA1 protein, Further, the model provides evidence that function and regulation of the human BRCA1 gene can be studied and manipulated in a genetically tractable mammalian system.

EDD, the human orthologue of the hyperplastic discs tumour suppressor gene, is amplified and overexpressed in cancer

EDD (E3 isolated by differential display), located at chromosome 8q22.3, is the human orthologue of the Drosophila melanogaster tumour suppressor gene 'hyperplastic discs' and encodes a HECT domain E3 ubiquitin protein-ligase. To investigate the possible involvement of EDD in human cancer, several cancers from diverse tissue sites were analysed for allelic gain or loss (allelic imbalance, AI) at the EDD locus using an EDD-specific microsatellite, CEDD, and other polymorphic microsatellites mapped in the vicinity of the 8q22.3 locus. Of 143 cancers studied, 38 had AI at CEDD (42% of 90 informative cases). In 14 of these cases, discrete regions of imbalance encompassing 8q22.3 were present, while the remainder had more extensive 8q aberrations. AI of CEDD was most frequent in ovarian cancer (22/47 informative cases, 47%), particularly in the serous subtype (16/22, 73%), but was rare in benign and borderline ovarian tumours. AI was also common in breast cancer (31%), hepatocellular carcinoma (46%), squamous cell carcinoma of the tongue (50%) and metastatic melanoma (18%). AI is likely to represent amplification of the EDD gene locus rather than loss of heterozygosity, as quantitative RT-PCR and immunohistochemistry showed that EDD mRNA and protein are frequently overexpressed in breast and ovarian cancers, while among breast cancer cell lines EDD overexpression and increased gene copy number were correlated. These results demonstrate that AI at the EDD locus is common in a diversity of carcinomas and that the EDD gene is frequently overexpressed in breast and ovarian cancer, implying a potential role in cancer progression.

Loss of heterozygosity of the APC gene in oral squamous cell carcinoma

The aim of this study was to investigate loss of heterozygosity (LOH) of the APC tumor suppressor gene loci, using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) in 40 cases of oral squamous cell carcinoma (OSCC). Observed informativity was 72.5% for APC exon 11 and 82.5% for APC exon 15. LOH at APC exon 11 was observed in 2 (6.9%) of 29 informative cases, and no LOH was observed for APC exon 15. Our results suggest that inactivation of the APC gene plays a minor role in the carcinogenesis of OSCC. (C) 2008 Elsevier GmbH. All rights reserved.

PTCH1 gene mutations in exon 17 and loss of heterozygosity on D9S180 microsatellite in sporadic and inherited human basal cell carcinomas

Background Basal cell carcinomas (BCCs) are the most frequent human cancer that results from malignant transformation of basal cells in the epidermis. Gorlin syndrome is a rare inherited autosomal dominant disease that predisposes with multiple BCCs and other birth defects. Both sporadic and inherited BCCs are associated with mutations in the tumor suppressor gene PTCH1, but there is still uncertainty on the role of its homolog PTCH2. Objectives To search for mutations and genomic instability in sporadic and inherited BCCs. Methods DNA obtained from leukocytes and tumor cells was amplified by polymerase chain reaction regarding five exons of PTCH1 and PTCH2 and neighboring microsatellites. Exons were sequenced and compared with the GenBank database. Results Only D9S180, of six microsatellites, showed loss of heterozygosity in three BCCs (two sporadic and one inherited). One sporadic BCC presented the mutation g. 2885G>C in exon 17 of PTCH1, which predicts the substitution p.R962T in an external domain of the protein. In addition, the leukocytes and tumor cells of one patient with Gorlin syndrome showed the mutation g. 2839T>G in the same exon and gene, which predicts a p.E947stop and truncated protein. All control and tumor samples presented IVS9 + 217T in intron 9 of PTCH1. Conclusion Mutations found in the PTCH1 gene and neighboring repetitive sequences may have contributed to the development of the studied BCCs.

Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan

Large, long-lived species experience more lifetime cell divisions and hence a greater risk of spontaneous tumor formation than smaller, short-lived species. Large, long-lived species are thus expected to evolve more elaborate tumor suppressor systems. In previous work, we showed that telomerase activity coevolves with body mass, but not lifespan, in rodents: telomerase activity is repressed in the somatic tissues of large rodent species but remains active in small ones. Without telomerase activity, the telomeres of replicating cells become progressively shorter until, at some critical length, cells stop dividing. Our findings therefore suggested that repression of telomerase activity mitigates the increased risk of cancer in larger-bodied species but not necessarily longer-lived ones. These findings imply that other tumor suppressor mechanisms must mitigate increased cancer risk in long-lived species. Here, we examined the proliferation of fibroblasts from 15 rodent species with diverse body sizes and lifespans. We show that, consistent with repressed telomerase activity, fibroblasts from large rodents undergo replicative senescence accompanied by telomere shortening and overexpression of p16(Ink4a) and p21(Cip1/Waf1) cycline-dependent kinase inhibitors. Interestingly, small rodents with different lifespans show a striking difference: cells from small shorter-lived species display continuous rapid proliferation, whereas cells from small long-lived species display continuous slow proliferation. We hypothesize that cells of small long-lived rodents, lacking replicative senescence, have evolved alternative tumor-suppressor mechanisms that prevent inappropriate cell division in vivo and slow cell growth in vitro. Thus, large-bodied species and small but long-lived species have evolved distinct tumor suppressor mechanisms.

TTYH2, a human homologue of the Drosophila melanogaster gene tweety, is located on 17q24 and upregulated in renal cell carcinoma

Using differential display PCR, we identified a novel gene upregulated in renal cell carcinoma. Characterization of the full-length cDNA and gene revealed that the encoded protein is a human homologue of the Drosophila melanogaster Tweety protein, and so we have termed the novel protein TTYH2. The orthologous mouse cDNA was also identified and the predicted mouse protein is 81% identical to the human protein. The encoded human TTYH2 protein is 534 amino acids and, like the other members of the tweety-related protein family, is a putative cell surface protein with five transmembrane regions. TTYH2 is located at 17q24; it is expressed most highly in brain and testis and at lower levels in heart, ovary, spleen, and peripheral blood leukocytes. Expression of this gene is upregulated in 13 of 16 (81%) renal cell carcinoma samples examined. In addition to a putative role in brain and testis, the overexpression of TTYH2 in renal cell carcinoma suggests that it may have an important role in kidney tumorigenesis.

A case of myoepithelial carcinoma displaying biallelic inactivation of the tumour suppressor gene APC in a patient with familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominant disorder caused by mutation of the APC gene. It is characterised by the appearance of hundreds to thousands of colorectal adenomas in adolescence and the subsequent development of colorectal cancer. Various extracolonic malignancies are associated with FAP, including desmoids and neoplasms of the stomach, duodenum, pancreas, liver, and brain. We present a family affected by FAP with an exon 14 APC mutation displaying two rare extracolonic lesions, a hepatoblastoma and a myoepithelial carcinoma. The hepatoblastoma was found in a male patient aged 2 years. The second lesion, a myoepithelial carcinoma of the right cheek, was found in a female patient aged 14 years. Inactivation of the normal APC allele was demonstrated in this lesion by loss of heterozygosity analysis, thus implicating APC in the initiation or progression of this neoplasm. This is the first reported case of this lesion in a family affected by FAP.