Effects of p53 mutations on apoptosis in mouse intestinal and human colonic adenomas

We have examined the effects of inactivation of the p53 tumor suppressor gene on the incidence of apoptotic cell death in two stages of the adenoma-to-carcinoma progression in the intestine: in early adenomas where p53 mutations are rare and in highly dysplastic adenomas where loss of p53 occurs frequently. Homozygosity for an inactivating germ-line mutation of p53 had no effect on the incidence or the rate of progression of ApcMin/+-induced adenomas in mice and also did not affect the frequency of apoptosis in the cells of these adenomas. To examine the effect of p53 loss on apoptosis in late-stage adenomas, we compared the incidence of apoptotic cell death before and after the appearance of highly dysplastic cells in human colonic adenomas. The appearance of highly dysplastic cells, which usually coincides during colon tumor progression with loss of heterozygosity at the p53 locus, did not correlate with a reduction in the incidence of apoptosis. These studies suggest that p53 is only one of the genes that determine the incidence of apoptotic in colon carcinomas and that wild-type p53 retards the progression of many benign colonic adenoma to malignant carcinomas by mechanism(s) other than the promotion of apoptosis.

A variant of DNA polymerase β acts as a dominant negative mutant

In eukaryotic cells, DNA polymerase β (polβ) carries out base-excision repair (BER) required for DNA maintenance, replication, recombination, and drug resistance. A specific deletion in one allele in the coding sequence of the polβ gene occurs in colorectal and breast carcinomas. The 87-bp deleted region encodes amino acid residues 208–236 in the catalytic domain of the enzyme. Here, we report evidence for expression of the wild-type (WT) and the truncated polβ proteins in colorectal tumors. To elucidate the potential functional consequences of polβ truncation, stable HeLa cell lines were established from cloned WT and variant polβΔ208–236. Cells expressing the variant protein exhibited substantially decreased BER activity. To test our hypothesis that truncated polβ may disrupt the function of the WT enzyme, we stably transfected mouse embryonic fibroblast 16.3 cells with polβΔ208–236 cDNA. Reverse transcription–PCR and Western blot analyses showed that the new cell line, 16.3ΔP, expresses the WT and the truncated polβ mRNA and proteins. BER and binding activities were undetectable in these cells. Furthermore, in vivo the 16.3ΔP cells were more sensitive to N-methyl-N′-nitro-N-nitrosoguanidine than the 16.3 cells. On adding increasing amounts of 16.3ΔP protein extracts, the BER and DNA binding activities of extracts of the parent 16.3 cell line progressively declined. These results strongly suggest that truncated polβ acts as a dominant negative mutant. The defective polβ may facilitate accumulation of mutations, leading to the expression of a mutator phenotype in tumor cells.

Loss of the gene encoding mannose 6-phosphate/insulin-like growth factor II receptor is an early event in liver carcinogenesis

This report shows that loss of heterozygosity at the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) locus occurred in 5/8 (63%) dysplastic liver lesions and 11/18 (61%) hepatocellular carcinomas (HCCs) associated with the high risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining allele were detected in 6/11 (55%) HCCs, including deletions in a polydeoxyguanosine region known to be a target of microsatellite instability. M6P/IGF2R allele loss was also found in cirrhotic tissue of clonal origin adjacent to these dysplastic lesions and HCCs, demonstrating that M6P/IGF2R inactivation occurs early in liver carcinogenesis. In conclusion, HCCs frequently develop from clonal expansions of phenotypically normal, M6P/IGF2R-mutated hepatocytes, providing further support for the idea that M6P/IGF2R functions as a liver tumor-suppressor gene.

Activating mutations for the Met tyrosine kinase receptor in human cancer

Recently, mutations in the Met tyrosine kinase receptor have been identified in both hereditary and sporadic forms of papillary renal carcinoma. We have introduced the corresponding mutations into the met cDNA and examined the effect of each mutation in biochemical and biological assays. We find that the Met mutants exhibit increased levels of tyrosine phosphorylation and enhanced kinase activity toward an exogenous substrate when compared with wild-type Met. Moreover, NIH 3T3 cells expressing mutant Met molecules form foci in vitro and are tumorigenic in nude mice. Enzymatic and biological differences were evident among the various mutants examined, and the somatic mutations were generally more active than those of germ-line origin. A strong correlation between the enzymatic and biological activity of the mutants was observed, indicating that tumorigenesis by Met is quantitatively related to its level of activation. These results demonstrate that the Met mutants originally identified in human papillary renal carcinoma are oncogenic and thus are likely to play a determinant role in this disease, and these results raise the possibility that activating Met mutations also may contribute to other human malignancies.

Proteinase inhibitors I and II from potatoes specifically block UV-induced activator protein-1 activation through a pathway that is independent of extracellular signal-regulated kinases, c-Jun N-terminal kinases, and P38 kinase

Solar UV irradiation is the causal factor for the increasing incidence of human skin carcinomas. The activation of the transcription factor activator protein-1 (AP-1) has been shown to be responsible for the tumor promoter action of UV light in mammalian cells. We demonstrate that proteinase inhibitor I (Inh I) and II (Inh II) from potato tubers, when applied to mouse epidermal JB6 cells, block UV-induced AP-1 activation. The inhibition appears to be specific for UV-induced signal transduction for AP-1 activation, because these inhibitors did not block UV-induced p53 activation nor did they exhibit any significant influence on epidermal growth factor-induced AP-1 transactivation. Furthermore, the inhibition of UV-induced AP-1 activity occurs through a pathway that is independent of extracellular signal-regulated kinases and c-Jun N-terminal kinases as well as P38 kinases. Considering the important role of AP-1 in tumor promotion, it is possible that blocking UV-induced AP-1 activity by Inh I or Inh II may be functionally linked to irradiation-induced cell transformation.

WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumors

Wnt family members are critical to many developmental processes, and components of the Wnt signaling pathway have been linked to tumorigenesis in familial and sporadic colon carcinomas. Here we report the identification of two genes, WISP-1 and WISP-2, that are up-regulated in the mouse mammary epithelial cell line C57MG transformed by Wnt-1, but not by Wnt-4. Together with a third related gene, WISP-3, these proteins define a subfamily of the connective tissue growth factor family. Two distinct systems demonstrated WISP induction to be associated with the expression of Wnt-1. These included (i) C57MG cells infected with a Wnt-1 retroviral vector or expressing Wnt-1 under the control of a tetracyline repressible promoter, and (ii) Wnt-1 transgenic mice. The WISP-1 gene was localized to human chromosome 8q24.1–8q24.3. WISP-1 genomic DNA was amplified in colon cancer cell lines and in human colon tumors and its RNA overexpressed (2- to >30-fold) in 84% of the tumors examined compared with patient-matched normal mucosa. WISP-3 mapped to chromosome 6q22–6q23 and also was overexpressed (4- to >40-fold) in 63% of the colon tumors analyzed. In contrast, WISP-2 mapped to human chromosome 20q12–20q13 and its DNA was amplified, but RNA expression was reduced (2- to >30-fold) in 79% of the tumors. These results suggest that the WISP genes may be downstream of Wnt-1 signaling and that aberrant levels of WISP expression in colon cancer may play a role in colon tumorigenesis.

A subset of skin tumor modifier loci determines survival time of tumor-bearing mice

Studies of mouse models of human cancer have established the existence of multiple tumor modifiers that influence parameters of cancer susceptibility such as tumor multiplicity, tumor size, or the probability of malignant progression. We have carried out an analysis of skin tumor susceptibility in interspecific Mus musculus/Mus spretus hybrid mice and have identified another seven loci showing either significant (six loci) or suggestive (one locus) linkage to tumor susceptibility or resistance. A specific search was carried out for skin tumor modifier loci associated with time of survival after development of a malignant tumor. A combination of resistance alleles at three markers [D6Mit15 (Skts12), D7Mit12 (Skts2), and D17Mit7 (Skts10)], all of which are close to or the same as loci associated with carcinoma incidence and/or papilloma multiplicity, is significantly associated with increased survival of mice with carcinomas, whereas the reverse combination of susceptibility alleles is significantly linked to early mortality caused by rapid carcinoma growth (χ2 = 25.22; P = 5.1 × 10−8). These data indicate that host genetic factors may be used to predict carcinoma growth rate and/or survival of individual backcross mice exposed to the same carcinogenic stimulus and suggest that mouse models may provide an approach to the identification of genetic modifiers of cancer survival in humans.

Binding of factor VIIa to tissue factor induces alterations in gene expression in human fibroblast cells: Up-regulation of poly(A) polymerase

Tissue factor (TF) is the cellular receptor for an activated form of clotting factor VII (VIIa) and the binding of factor VII(a) to TF initiates the coagulation cascade. Sequence and structural patterns extracted from a global alignment of TF confers homology with interferon receptors of the cytokine receptor super family. Several recent studies suggested that TF could function as a genuine signal transducing receptor. However, it is unknown which biological function(s) of cells are altered upon the ligand, VIIa, binding to TF. In the present study, we examined the effect of VIIa binding to cell surface TF on cellular gene expression in fibroblasts. Differential mRNA display PCR technique was used to identify transcriptional changes in fibroblasts upon VIIa binding to TF. The display showed that VIIa binding to TF either up or down-regulated several mRNA species. The differential expression of one such transcript, VIIa-induced up-regulation, was confirmed by Northern blot analysis. Isolation of a full-length cDNA corresponding to the differentially expressed transcript revealed that VIIa-up-regulated gene was poly(A) polymerase. Northern blot analysis of various carcinomas and normal human tissues revealed an over expression of PAP in cancer tissues. Enhanced expression of PAP upon VIIa binding to tumor cell TF may potentially play an important role in tumor metastasis.

Identification of semaphorin E as a non-MDR drug resistance gene of human cancers

To improve cancer chemotherapy, a better understanding of the molecular mechanisms of drug resistance is essential. To identify the molecules responsible for drug resistance that is unrelated to MDR1 or MRP gene products, a eukaryotic expression cDNA library of cis-diamminedichloroplatinum(II) (CDDP)-resistant ovarian cancer TYKnuR cells was introduced into Cos-7 cells. After repeated CDDP selection, cDNA homologous to murine semaphorin E was isolated from surviving cells. Human semaphorin E (H-sema E) was overexpressed in CDDP-resistant cell lines and was readily induced not only by diverse chemotherapeutic drugs but also by x-ray and UV irradiation. Transfection of H-sema E conferred a drug-resistant phenotype to CDDP-sensitive cells. In addition, the aberrant expression of H-sema E protein was detected immunohistochemically in 14 of 42 (33.3%) recurrent squamous cell carcinomas removed at autopsy after extensive radiochemotherapy. Recently, another member of the semaphorin family, CD100, was shown to significantly improve the viability of B lymphocytes. These results suggest the involvement of semaphorins in diverse cell survival mechanisms.

Gain of imprinting at chromosome 11p15: A pathogenetic mechanism identified in human hepatocarcinomas

Genomic imprinting is a reversible condition that causes parental-specific silencing of maternally or paternally inherited genes. Analysis of DNA and RNA from 52 human hepatocarcinoma samples revealed abnormal imprinting of genes located at chromosome 11p15 in 51% of 37 informative samples. The most frequently detected abnormality was gain of imprinting, which led to loss of expression of genes present on the maternal chromosome. As compared with matched normal liver tissue, hepatocellular carcinomas showed extinction or significant reduction of expression of one of the alleles of the CDKN1C, SLC22A1L, and IGF2 genes. Loss of maternal-specific methylation at the KvDMR1 locus in hepatocarcinoma correlated with abnormal expression of CDKN1C and IGF2, suggesting a function for KvDMR1 as a long-range imprinting center active in adult tissues. These results point to the role of epigenetic mechanisms leading to loss of expression of imprinted genes at chromosome region 11p15 in human tumors.

Cure of human carcinoma xenografts by a single dose of pretargeted yttrium-90 with negligible toxicity

A covalent conjugate (NR-LU-10/SA) was prepared between streptavidin (SA) and NR-LU-10, a mAb that binds an antigen expressed on the surface of most human carcinomas. NR-LU-10/SA was injected into nude mice bearing human tumor xenografts. Injection of biotinylated galactosyl-human serum albumin reduced the circulating levels of conjugate by 95%. Subsequent administration of 90Y-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin achieved peak uptake at the tumor within 2 hr while >80% of the radioactivity was eliminated in the urine. A single dose of 600–800 μCi of 90Y-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin produced cures in 10/10 mice with established (>200 mm3) s.c. human small cell lung or colon cancer xenografts and 8/10 cures in mice with human breast cancer xenografts without significant toxicity.

Chromosomal instability and cytoskeletal defects in oral cancer cells

Oral squamous cell carcinomas are characterized by complex, often near-triploid karyotypes with structural and numerical variations superimposed on the initial clonal chromosomal alterations. We used immunohistochemistry combined with classical cytogenetic analysis and spectral karyotyping to investigate the chromosomal segregation defects in cultured oral squamous cell carcinoma cells. During division, these cells frequently exhibit lagging chromosomes at both metaphase and anaphase, suggesting defects in the mitotic apparatus or kinetochore. Dicentric anaphase chromatin bridges and structurally altered chromosomes with consistent long arms and variable short arms, as well as the presence of gene amplification, suggested the occurrence of breakage–fusion–bridge cycles. Some anaphase bridges were observed to persist into telophase, resulting in chromosomal exclusion from the reforming nucleus and micronucleus formation. Multipolar spindles were found to various degrees in the oral squamous cell carcinoma lines. In the multipolar spindles, the poles demonstrated different levels of chromosomal capture and alignment, indicating functional differences between the poles. Some spindle poles showed premature splitting of centrosomal material, a precursor to full separation of the microtubule organizing centers. These results indicate that some of the chromosomal instability observed within these cancer cells might be the result of cytoskeletal defects and breakage–fusion–bridge cycles.

Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization

The development of skin carcinomas presently is believed to be correlated with mutations in the p53 tumor suppressor and ras gene as well as with the loss of chromosome 9. We now demonstrate that, in addition, loss of chromosome 15 may be a relevant genetic defect. Reintroduction of an extra copy of chromosome 15, but not chromosome 4, into the human skin carcinoma SCL-I cells, lacking one copy of each chromosome, resulted in tumor suppression after s.c. injection in mice. Transfection with thrombospondin-1 (TSP-1), mapped to 15q15, induced the same tumor suppression without affecting cell proliferation in vitro or in vivo. Halted tumors remained as small cysts encapsulated by surrounding stroma and blood vessels. These cysts were characterized by increased TSP-1 matrix deposition at the tumor/stroma border and a complete lack of tumor vascularization. Coinjection of TSP-1 antisense oligonucleotides drastically reduced TSP-1 expression and almost completely abolished matrix deposition at the tumor/stroma border. As a consequence, the tumor phenotype reverted to a well vascularized, progressively expanding, solid carcinoma indistinguishable from that induced by the untransfected SCL-I cells. Thus, these data strongly suggest TSP-1 as a potential tumor suppressor on chromosome 15. The data further propose an unexpected mechanism of TSP-1-mediated tumor suppression. Instead of interfering with angiogenesis in general, in this system TSP-1 acts as a matrix barrier at the tumor/stroma border, which, by halting tumor vascularization, prevents tumor cell invasion and, thus, tumor expansion.

Immune surveillance against a solid tumor fails because of immunological ignorance

Many peripheral solid tumors such as sarcomas and carcinomas express tumor-specific antigens that can serve as targets for immune effector T cells. Nevertheless, overall immune surveillance against such tumors seems relatively inefficient. We studied immune surveillance against a s.c. sarcoma expressing a characterized viral tumor antigen. Surprisingly, the tumor cells were capable of inducing a protective cytotoxic T cell response if transferred as a single-cell suspension. However, if they were transplanted as small tumor pieces, tumors readily grew. Tumor growth correlated strictly with (i) failure of tumor cells to reach the draining lymph nodes and (ii) absence of primed cytotoxic T cells. Cytotoxic T cells were not tolerant or deleted because a tumor antigen-specific cytotoxic T cell response was readily induced in lymphoid tissue by immunization with virus or with tumor cells even in the presence of large tumors. Established tumors were rejected by vaccine-induced effector T cells if effector T cells were maintained by prolonged or repetitive vaccination, but not by single-dose vaccination. Thus, in addition to several other tumor-promoting parameters, some antigenic peripheral sarcomas—and probably carcinomas—may grow not because they anergize or tolerize tumor-specific T cells, but because such tumors are immunologically dealt with as if they were in a so-called immunologically privileged site and are ignored for too long.

E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium

Ovarian carcinomas are thought to arise in the ovarian surface epithelium (OSE). Although this tissue forms a simple epithelial covering on the ovarian surface, OSE cells exhibit some mesenchymal characteristics and contain little or no E-cadherin. However, E-cadherin is present in metaplastic OSE cells that resemble the more complex epithelia of the oviduct, endometrium and endocervix, and in primary epithelial ovarian carcinomas. To determine whether E-cadherin was a cause or consequence of OSE metaplasia, we expressed this cell-adhesion molecule in simian virus 40-immortalized OSE cells. In these cells the exogenous E-cadherin, all three catenins, and F-actin localized at sites of cell–cell contact, indicating the formation of functional adherens junctions. Unlike the parent OSE cell line, which had undergone a typical mesenchymal transformation in culture, E-cadherin-expressing cells contained cytokeratins and the tight-junction protein occludin. They also formed cobblestone monolayers in two-dimensional culture and simple epithelia in three-dimensional culture that produced CA125 and shed it into the culture medium. CA125 is a normal epithelial-differentiation product of the oviduct, endometrium, and endocervix, but not of normal OSE. It is also a tumor antigen that is produced by ovarian neoplasms and by metaplastic OSE. Thus, E-cadherin restored some normal characteristics of OSE, such as keratin, and it also induced epithelial-differentiation markers associated with weakly preneoplastic, metaplastic OSE and OSE-derived primary carcinomas. The results suggest an unexpected role for E-cadherin in ovarian neoplastic progression.