p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells.
Cayrol C. Knibiehler M. Ducommun B.
Institut de Pharmacologie et de Biologie Structurale du CNRS Universite Paul Sabatier, Toulouse, France.
A unique feature of p21 that distinguishes it from the other cyclin-dependent kinase (CDK) inhibitors is its ability to associate with the proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerases delta and epsilon. While it is now well established that inhibition of cyclin/CDK complexes by p21 can result in G1 cell cycle arrest, the consequences of p21/PCNA interaction on cell cycle progression have not yet been determined. Here, we show, using a tetracycline-regulated system, that expression of wild-type p21 in p53-deficient DLD1 human colon cancer cells inhibits DNA synthesis and causes G1 and G2 cell cycle arrest. Similar effects are observed in cells expressing p21CDK-, a mutant impaired in the interaction with CDKs, but not in cells expressing p21PCNA-, a mutant deficient for the interaction with PCNA. Analysis of cells treated with a p21-derived PCNA-binding peptide provides additional evidence that the growth inhibitory effects of p21 and p21CDK result from their ability to bind to PCNA. Our results suggest that p21 might inhibit cell cycle progression by two independent mechanisms, inhibition of cyclin/CDK complexes, and inhibition of PCNA function resulting in both G1 and G2 arrest.
The plasminogen activator system in pancreas cancer: role of t-PA in the invasive potential in vitro.
Paciucci R. Tora M. Diaz VM. Real FX.
Unitat de Biologia Cellular i Molecular, Institut Municipal d'Investigacio Medica, Barcelona, Spain.
Plasminogen activators (PAs) play an important role in tumor cell invasion. We have analysed the expression of tissue-type PA (t-PA), urokinase-type PA (u-PA), and their respective receptors, annexin II and u-PAR, in normal and neoplastic cultures of pancreatic cells, as well as in pancreatic tissues, and have examined their role in tumor invasiveness in vitro. Using Northern blotting, Western blotting, and ELISA, t-PA is detected in cultured pancreas cancer cells displaying a well differentiated phenotype but it is undetectable in less differentiated cells and in normal pancreatic cultures. In contrast, u-PA transcripts, protein, and enzymatic activity are detected both in cancer cells and in normal cultures. Higher levels of u-PAR and annexin II are present in cancer cells than in normal cultures and, in SK-PC-1 cells, both receptors are localized in the basolateral membrane. In vitro invasion assays indicate that both t-PA and u-PA contribute to the invasiveness of SK-PC-1 cells through reconstituted extracellular matrix. To determine the relevance of these studies to pancreas cancer, immunohistochemical assays have been used to examine the expression of t-PA, u-PA, and their receptors in normal and neoplastic tissues. t-PA is absent from normal pancreas and from tumor associated pancreatitis, whereas it is detected in the majority of pancreas cancer tissues (16/17). Annexin II is also overexpressed in some tumors (5/13). u-PAR is overexpressed in most tumor samples examined (14/15), while u-PA is weakly detected in a low number of cases (3/14); both u-PAR and u-PA are overexpressed in areas of tumor associated pancreatitis. Indirect evidences indicate that K-ras and p53 mutated proteins can regulate the expression of PAs. In pancreatic cancer we have found an association between codon 12 K-ras mutations and t-PA expression (P=0.04). These results support the contention that, in the exocrine pancreas, activation of t-PA is more specifically associated to neoplastic transformation and to the invasive phenotype, whereas the induction of u-PA/u-PAR system might be more relevant to inflammatory or non-neoplastic events.
Frequent homozygous deletions in the FRA3B region in tumor cell lines still leave the FHIT exons intact.
Wang L. Darling J. Zhang JS. Qian CP. Hartmann L. Conover C. Jenkins R. Smith DI.
Department of Laboratory Medicine and Pathology, Mayo Clinic/Foundation, Rochester, Maine 55902, USA.
FRA3B at human chromosomal band 3p14.2 is the most active common fragile site in the human genome. The molecular mechanism of fragility at this region remains unknown but does not involve expansion of a trinucleotide or minisatellite repeat as has been observed for several of the cloned rare fragile sites. Deletions and rearrangements at FRA3B have been observed in a number of distinct tumors. The recently identified putative tumor suppressor gene FHIT spans FRA3B, and various groups have reported identifying deletions in this gene in different tumors. Using a high density of PCR amplifiable markers within FRA3B searching for deletions in the FRA3B region, we have analysed 21 tumor cell lines derived from renal cell, pancreatic, and ovarian carcinomas. We found a commonly deleted region in the renal cell and ovarian carcinoma cell lines located in the middle of an HPV16 viral integration site. Despite the presence of deletions in the FRA3B region in most of the cell lines, we did not detect alterations in FHIT exons in any of the cell lines examined. Thus, deletions of 3p14.2 in these carcinoma cell lines may simply reflect instability of the FRA3B region during tumor progression.
Intestinal cancer in patients with a germline mutation in the down-regulated in adenoma (DRA) gene.
Hemminki A. Hoglund P. Pukkala E. Salovaara R. Jarvinen H. Norio R. Aaltonen LA.
Department of Medical Genetics, Haartman Institute, University of Helsinki, Finland.
A recent study has revealed that germline mutations of the down-regulated in adenoma (DRA) gene are a likely cause of a recessive intestinal absorption defect, congenital chloride diarrhea. This finding was in accordance with previous works showing that DRA encodes a sodium independent transporter for sulfate and oxalate. Although DRA was originally reported as a candidate tumor suppressor, these studies have questioned the relevance of DRA in cancer. To evaluate whether further studies on the role of DRA in tumorigenesis are still of interest, we examined whether individuals carrying germline DRA mutations have an excess of intestinal cancer. Cancer status of 229 members of 36 Finnish congenital chloride diarrhea families (44 homozygous patients, 70 heterozygous parents, and 115 grandparents at 50% risk of being a DRA mutation carrier) was checked at the Finnish Cancer Registry and the risk of intestinal cancer was found slightly elevated (standardized incidence ratio 3.4, 95% confidence interval 1.4-7.0, P < 0.05). While this result does not unambiguously demonstrate an increased intestinal cancer risk in DRA mutation carriers, it should promote further studies to determine the possible role of DRA in cancer.
Mutated p21(WAF1/CIP1/SDI1) lacking CDK-inhibitory activity fails to prevent apoptosis in human colorectal carcinoma cells.
Lu Y. Yamagishi N. Yagi T. Takebe H.
Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Japan.
Human colorectal tumor cell lines were established which express wildtype p21 or p21 with a mutation at codon 46 (Cys) or 140 (Gly) on IPTG treatment (LacSwitch). The IPTG-induced wildtype p21 bound to CDK2 and PCNA and inhibited CDK activity in the cells and reduced cell growth rate; whereas, both IPTG-induced mutated p21 proteins neither bound to CDK2 nor affected the CDK activity but did bind to PCNA, and they did not affect the cell growth rate. Wildtype p21 suppressed apoptosis and enhanced survival of X-ray-irradiated or adriamycin-treated cells; but, mutated p21 neither suppressed apoptosis nor affected cell survival. When cells were treated with mimosine, a p53-independent p21-inducer, or butyrolactone I, a specific inhibitor of CDK, cellular endogenous p21 was induced and X-ray or adriamycin-induced apoptosis was blocked. These results suggest that CDK-binding or CDK-inhibitory activity of p21 is required to prevent apoptosis, i.e., CDK is required for apoptosis in human tumor cells.
Suppression of invasive properties of colon cancer cells by a metastasis suppressor KAI1 gene.
Takaoka A. Hinoda Y. Satoh S. Adachi Y. Itoh F. Adachi M. Imai K.
First Department of Internal Medicine, Sapporo Medical University, Japan.
KAI1 is a potential metastatic suppressor gene for prostate cancer. We found by Northern blot analysis that six of ten (60%) gastric and colon cancer cell lines exhibited undetectable or very low expression level of KAI1 mRNA. The effects of KAI1 on the adhesion, motility and invasiveness of colon cancer cells was therefore investigated by using two kinds of stable transfectants, i.e., antisense transfectants of BM314 cells whose KAI1 mRNA expression was suppressed by transfer of antisense KAI1 cDNA and sense transfectants of DLD-1 cells with the enhanced KAI1 mRNA by sense cDNA transfer. The following results were obtained: (1) KAI1 gene expression had no significant effect on in vitro cell growth rate of colon cancer BM314 and DLD-1 cells; (2) Cell aggregation assay showed that KAI1 enhanced the Ca++-independent aggregatability of those colon cancer cells; (3) It was revealed by cell motility and invasion assays that KAI1 suppressed both the motility and in vitro invasiveness of those cells and (4) Furthermore, both the binding to fibronectin and the migration on fibronectin-coated plates of those cells were inhibited by KAI1 expression. These suggest that reduced KAI1 gene expression may contribute to the invasiveness and metastatic ability of colon cancer cells.
Increased transversions in a novel mutator colon cancer cell line.
Eshleman JR. Donover PS. Littman SJ. Swinler SE. Li GM. Lutterbaugh JD. Willson JK. Modrich P. Sedwick WD. Markowitz SD. Veigl ML.
Department of Pathology, University Hospitals of Cleveland and Case Western Reserve University, Ohio 44106, USA.
We describe a novel mutator phenotype in the Vaco411 colon cancer cell line which increases the spontaneous mutation rate 10-100-fold over background. This mutator results primarily in transversion base substitutions which are found infrequently in repair competent cells. Of the four possible types of transversions, only three were principally recovered. Spontaneous mutations recovered also included transitions and large deletions, but very few frameshifts were recovered. When compared to known mismatch repair defective colon cancer mutators, the distribution of mutations in Vaco411 is significantly different. Consistent with this difference, Vaco411 extracts are proficient in assays of mismatch repair. The Vaco411 mutator appears to be novel, and is not an obvious human homologue of any of the previously characterized bacterial or yeast transversion phenotypes. Several hypotheses by which this mutator may produce transversions are presented.
Association of an 80 kDa protein with C-CAM1 cytoplasmic domain correlates with C-CAM1-mediated growth inhibition.
Luo W. Earley K. Tantingco V. Hixson DC. Liang TC. Lin SH.
Department of Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston 77030, USA.
Decreased expression of C-CAM, a member of the CEA family of immunoglobulin like cell adhesion molecules, occurs in carcinomas of the colon, liver and prostate. Down regulation of C-CAM during the early stages of carcinogenesis in rat liver and human prostate has also been reported. We have recently shown that restoration of the expression of the isoform with long cytoplasmic domain, C-CAM1, leads to suppression of the tumorigenicity of prostatic carcinoma cells in vivo and growth suppression in vitro. These observations suggest that C-CAM1 may play an important role in regulating cell growth in normal tissues. Previous studies have demonstrated that the function of many members of the Ig-supergene family is dependent on interactions with cytoplasmic proteins. In the present study, we have used a bifunctional cross-linker to identify cellular proteins that interact directly with C-CAM1. Immunoblot analysis of WGA bound membrane proteins crosslinked with DSS identified a 180 kDa complex composed of C-CAM and an 80 kDa protein designated CAP-80 (C-CAM Associated Protein). Immunoprecipitation with anti-C-CAM antibodies showed that CAP-80 was co-precipitated with C-CAM from detergent solubilized, WGA-purified proteins. To assess the specificity of CAP-80 binding, the ability of CAP-80 to form stable complexes with C-CAM1 mutants expressed in insect cells was tested. Deletion of the cytoplasmic domain of C-CAM1 abolished complex formation whereas deletion of the extracellular Ig domains had no effect. These results suggest that a CAP-80 homologue (ICAP-80) is present in insect cells and ICAP-80 interacts with the cytoplasmic domain of C-CAM1. Replacement of Tyr488, a residue in the cytoplasmic domain known to be phosphorylated in vivo, with Phe did not diminish the association between C-CAM1 and ICAP-80, suggesting that Tyr488 phosphorylation is not required for association. The ability of various C-CAM1 mutants to associate with ICAP-80 correlated with their growth inhibitory activities, suggesting that ICAP-80/CAP-80 may play an important role in C-CAM1-mediated growth inhibition.
Nuclear factor - kappaB-dependent regulation of p53 gene expression induced by daunomycin genotoxic drug.
Hellin AC. Calmant P. Gielen J. Bours V. Merville MP.
Laboratory of Medical Chemistry, University of Liege, Belgium.
Anthracycline drugs are widely used for the treatment of solid tumors and leukemia, but the molecular basis of their biological effect is still poorly understood. In the HCT116 colon carcinoma cell line, which retains a wild-type inducible p53 gene, we show that the anthracycline daunomycin is a potent inducer of p53 and NF-kappaB transcription factors. Nuclear accumulation of p53 protein occurred because of increased protein stability and enhanced gene expression. In addition, daunomycin induced the p53 promoter through the binding of p50/p65 NF-kappaB heterodimers to the kappaB site in the p53 promoter. Under our conditions, the free radical scavengers NAC and PDTC were not able to block NF-kappaB activation or p53 induction, indicating that reactive oxygen intermediates were not involved in the cellular response to daunomycin stimulation. Overexpression of a stable unresponsive IkappaBalpha mutant in HCT116 cells resulted in a complete inhibition of the NF-kappaB activation but only a partial impairment of the p53 protein accumulation induced by daunomycin. We conclude that the p53-activating signal generated by daunomycin is partially regulated by NF-kappaB.
Mismatch repair deficiency leads to a unique mode of colorectal tumorigenesis characterized by intratumoral heterogeneity.
Habano W. Sugai T. Nakamura S.
Division of Pathology, School of Medicine, Iwate Medical University, Morioka, Japan.
In order to determine the effects of mismatch repair (MMR) deficiencies in sporadic colorectal carcinomas, 45 such cancers were examined using a sensitive method called crypt isolation technique. Loss of heterozygosity (LOH) in the MSH2 or MLH1 gene was more frequently observed in replication error (RER) (+) carcinomas than in RER (-) carcinomas, which implied that loss of one normal allele could partly affect repair capacity. MSH2 gene defects at both alleles were observed in two carcinomas, which showed severe repair deficiencies. Interestingly, unlike the situation observed in the p53 gene, the MSH2 and MLH1 genes did not show complete LOH. Novel crypt isolation-based subpopulation (CISP) analysis demonstrated that at least two distinct carcinoma subpopulations existed in most carcinomas that showed incomplete LOH; one with and one without LOH. In one carcinoma that had germline mutation and somatic incomplete LOH of the MSH2 gene, the mutator phenotype was only observed in populations affected in both alleles. Thus, the MSH2 gene appears to possess the two hits mechanism of tumor suppressor genes. However, unlike the tumor suppressor genes, MMR gene defects lead to a unique mode of colorectal tumorigenesis characterized by intratumoral heterogeneity.
Human smooth muscle alpha-actin gene is a transcriptional target of the p53 tumor suppressor protein.
Comer KA. Dennis PA. Armstrong L. Catino JJ. Kastan MB. Kumar CC.
Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
Smooth muscle (sm) alpha-actin is expressed in vascular smooth muscle cells and fibroblast cells. Its expression is regulated by cell proliferation and repressed during oncogenic transformation. In this study, we demonstrate that p53 activation is associated with a dramatic increase in organized microfilament bundles and an increase in sm alpha-actin mRNA level. Wild-type p53, but not mutant p53, strongly stimulated human sm alpha-actin promoter activity in p53 null cell lines. The sequences homologous to the p53 consensus sequence and to the p53 binding sequence from the muscle creatine kinase, were found within a specific region of the sm alpha-actin promoter. This sequence was sufficient to confer p53-dependent activation to a heterologous promoter and p53 was capable of binding to this sequence as assessed by gel shift analysis. Ionizing irradiation of colorectal tumor cells caused an increase in alpha-actin mRNA level in a p53-dependent manner. Taken together, these results demonstrate that human sm alpha-actin gene is a transcriptional target for p53 tumor suppressor protein and represents the first example of a cytoskeletal gene with a functionally defined p53 response element.
Molecular analysis of the IL-2 receptor beta chain gene expressed in human tumor cells.
Suminami Y. Kashii Y. Law JC. Lin WC. Stanson J. Reichert TE. Rabinowich H. Whiteside TL.
The University of Pittsburgh Cancer Institute and Department of Pathology, University of Pittsburgh School of Medicine, Pennsylvania 15213, USA.
Interleukin-2 (IL-2) is recognized as a T cell growth factor. We have previously reported that human carcinoma cell lines are inhibited in growth by exogenous IL-2, which binds to the IL-2 receptor beta (IL-2Rbeta) chain ubiquitously expressed on the surface of tumor cells. A possibility was considered that IL-2Rbeta on carcinomas responsible for negative signaling was different from that expressed on hematopoietic cells. To investigate this possibility, mRNA for the IL-2Rbeta chain was amplified and compared in carcinoma and lymphoid cells. Using RT-PCR with pairs of sense-antisense oligonucleotide primers specific for the various regions of extracellular, transmembrane and intracellular domains of the IL-2Rbeta chain, we amplified mRNA obtained from three human carcinoma cell lines and human lymphoid cells as controls. The identity of the amplicons was confirmed by Southern analysis with the 32P-labeled cDNA probe coding for the entire span of the IL-2Rbeta chain. In addition, genomic DNA obtained from the tumor cell lines was sequenced to examine the possibility that a mutation is present in the gene coding for the intracellular IL-2Rbeta chain domain. No mutations or deletions were detected. The message for all three domains of the beta chain was identical in tumor cells and in normal lymphoid cells used as controls. Also, by Western blot and northern analyses no differences between IL-2Rbeta chain in tumors vs that expressed in lymphoid cells were demonstrable. The IL-2Rgamma chain, which participates in IL-2/IL-2R signaling pathway, was expressed in tumor cells. Expression of JAK1 transcripts in these cells was comparable to that in lymphocytes. However, RT-PCR analysis identified differences in expression of JAK3 splice variants (B and M) in tumor cells. These differences may be responsible for altered downstream signaling by IL-2. Overall, our data indicate that the same IL-2/IL-2R pathway is operative in human carcinomas and in normal epithelial or lymphoid cells.
Stable reintroduction of wild-type P53 (MTmp53ts) causes the induction of apoptosis and neuroendocrine-like differentiation in human ductal pancreatic carcinoma cells.
Lang D. Miknyoczki SJ. Huang L. Ruggeri BA.
Department of Pathology and Laboratory Medicine, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102, USA.
Pancreatic ductal adenocarcinoma is one of the major causes of cancer mortality in the industrialized world, having among the poorest prognosis of any malignancy. Mutations or alterations in the p53 tumor suppressor gene/protein are observed in 50-70% of these cancers, yet little information is available regarding the phenotypic effects of restoration of wild-type (wt) p53 function in pancreatic ductal carcinoma cells. The consequences of stable reintroduction of wt p53 on apoptosis and differentiation was examined in a poorly differentiated pancreatic carcinoma cell line (Panc-1), possessing only mutant (mt) p53 (codon 273 mutation). Cells were transfected with a temperature-sensitive mouse p53val135 (tsp53) vector under additional control of a genetically-modified metallothionein promoter. This tsp53 has a 'mt' phenotype at 37.5 degrees C, and a 'wt' phenotype at 32.5 degrees C and the presence of 100 microM ZnCl2. Stable expression of wt p53 caused upregulation of the p21/WAF1 gene, and G1 growth arrest as shown by flow cytometry and BrdU labeling. Additionally, apoptosis was induced 8-12 post-induction in the majority of the cells (60-70%), as demonstrated by morphological changes, in situ TdT labeling and internucleosomal laddering. However, a subpopulation (30%) of the transfectants survived this apoptotic fate. Unlike the epithelial parental Panc-1 cells, these cells exhibited the appearance of a neuroendocrine-like phenotype with extensive branch-like processes, and marked cytoplasmic and cytoskeletal immunostaining for tau-2, synaptophysin, and chromogranin A. These studies suggest that stable and regulated expression of wt p53 can have multiple phenotypic consequences (apoptosis and altered differentiation to a neuroendocrine-like phenotype) in poorly-differentiated pancreatic carcinoma cells.
Potential role for cathepsin D in p53-dependent tumor suppression and chemosensitivity.
Wu GS. Saftig P. Peters C. El-Deiry WS.
Howard Hughes Medical Institute, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Cathepsin D (CD), the major intracellular aspartyl protease, is a mediator of IFN-gamma and TNF-alpha induced apoptosis. Using subtractive hybridization screening we isolated CD as an upregulated transcript in PA1 human ovarian cancer cells undergoing adriamycin-induced apoptosis. CD mRNA levels increased in wild-type p53-expressing PA1, ML1 leukemia and U1752 lung cancer cells but not in mutant p53-expressing cells following adriamycin exposure. Overexpression of CD inhibited growth of colon, liver, and ovarian cancer cells. CD protein expression was increased by exposure of ML1 cells to etoposide, adriamycin or gamma-radiation. Inhibition of CD protease with Pepstatin A suppressed p53-dependent apoptosis in lymphoid cells, suggesting a possible role for CD in p53-dependent cell death. CD-/- fibroblasts were found to be more resistant to killing by adriamycin and etoposide, as compared to CD+/+ cells. Two p53 DNA-binding sites located in the CD-promoter specifically bound to p53 protein in vitro and appeared to mediate transactivation of a CD-promoter luciferase-reporter during p53-dependent apoptosis. These observations link CD protease to p53-dependent tumor suppression and chemosensitivity.
The human hepatitis B virus transactivator X gene product regulates Sp1 mediated transcription of an insulin-like growth factor II promoter 4.
Lee YI. Lee S. Lee Y. Bong YS. Hyun SW. Yoo YD. Kim SJ. Kim YW. Poo HR.
Molecular Cell Biology Research Division, Korea Research Institute of Bioscience and Biotechnology, Korea Institute of Science and Technology, Taejon.
Human hepatitis B virus (HBV) is one of the causative agents of hepatocellular carcinoma (HCC). The virus encodes a 17 kDa protein, X, which is known to be a causative agent in the formation of HCC. An insulin-like growth factor-II (IGF-II) is expressed during the formation of HCC. Among the four promoters of the IGF-II gene, promoters 2, 3 and 4 become activated during the formation of HCC. The high frequency of detection of hepatitis B virus X (HBV-X) antigen in liver cells from patients with chronic hepatitis, cirrhosis, and liver cancer suggested that the expressions of HBV-X and IGF-II are associated. Studies were carried out to test the relationship between the HBV-X gene product and the activation of IGF-II promoter 4. We demonstrated that the HBV-X protein increases the endogenous IGF-II expression from promoter 3 and 4 of IGF-II gene. Analysis of the fourth promoter of IGF-II gene showed that the HBV-X gene product positively regulates transcription. Two copies of a motif are responsible for conferring HBV-X regulation on the fourth promoter of IGF-II. These motifs have been identified as Sp1 binding sites. Sp1 binding to IGF-II P4 promoter was identified by gel mobility shift assay using purified Sp1. By using a GAL4-Sp1 fusion protein it was demonstrated that HBV-X positively regulates the Spl mediated transcriptional activity of IGF-II in vivo. A protein-affinity chromatography experiment showed that HBV-X protein does not bind directly to Sp1, but HBV-X does augment the DNA binding activity of the phosphorylated form of Sp1 in HepG2 cells. Sp1 was phosphorylated by HBV-X and its DNA-binding activity was up-regulated upon HBV-X transfections. Various HBV-X mutant expression vectors were used for the demonstration of specific interactions between Sp1 and HBV-X. These results indicate that HBV-X functions as a positive regulator of transcription, and that Sp1 is a direct target for the transcriptional regulation of IGF-II. Increasing the DNA binding ability of the phosphorylated form of Sp1 by HBV-X might be an important mechanism for regulating the IGF-II gene expression and possibly promoting cell division during hepatic carcinogenesis. Our experimental results suggest that expression of HBV-X might induce the expression of IGF-II and the IGF-II might play a role in hepatitis B virus pathogenesis during the formation of HCC.
Microsatellite mutation rates in cancer cell lines deficient or proficient in mismatch repair.
Hanford MG. Rushton BC. Gowen LC. Farber RA.
Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 27599, USA.
A selectable system has been used to determine mutation rates within a microsatellite sequence in human cancer cell lines with or without defects in mismatch repair. A sequence consisting of 17 repeats of poly (dC-dA).poly(dT-dG) [abbreviated as (Ca)17] was inserted near the 5' end of the bacterial neomycin-resistance gene in a plasmid vector, such that the reading frame of the neo gene is disrupted. This plasmid was introduced into cancer cell lines, where it became integrated into the cellular genome. Clones with insertions or deletions of CA-repeats that restored the normal reading frame of the neo gene were selected in G418, and mutation rates were determined by fluctuation analysis. The rates of reversion in LoVo cells, which are deficient for hMSH2, were about one in a thousand per generation, which is approximately two orders of magnitude higher than in the repair-proficient HT-1080 human fibrosarcoma cell line. The mutation rates in H6 cells, which are derived from the hMLH1-deficient HCT116 line, were more heterogeneous than in LoVo, but all were considerably higher than in the repair-proficient line. Nearly all of the revertants of the repair-deficient lines had deletions of a single CA-repeat from the microsatellite sequence, whereas repair-proficient cells had a broader spectrum of mutations.
Prolonged cell survival enhances peritoneal dissemination of gastric cancer cells.
Yawata A. Adachi M. Okuda H. Naishiro Y. Takamura T. Hareyama M. Takayama S. Reed JC. Imai K.
The First Department of Internal Medicine, Sapporo Medical University School of Medicine, Japan.
Bcl-2 and a Bcl-2-binding protein BAG-1 function in protection from apoptosis induced by a variety of stimuli. Deregulated expression of Bcl-2 leads to inhibition of apoptosis and is correlated with development of various cancers. Here, we provide evidence that prolonged cell survival introduced by overproduction of Bcl-2 or BAG-1 strongly enhances peritoneal dissemination of human gastric cancer MKN74 cells. Gene transfer-mediated overexpression of Bcl-2 or BAG-1 led to prolonged cell survival of MKN74 cells against serum-starved apoptosis and anoikis. When the viable transfectants were inoculated into the intraperitoneal cavity of BALB/c nude mice, the Bcl-2-expressing MKN74 cells and the BAG-1-expressing MKN74 cells exhibited strongly enhanced peritoneal dissemination in BALB/c nude mice and whole disseminated tumor weights were increased by 4-fold and 3.3-fold, respectively, compared with the control transfectants. The enhanced peritoneal dissemination of MKN74-Bcl-2 and MKN74-BAG-1 transfectants correlated well with resistance to cell death induced by serum-starvation and anoikis. However, the overexpression of Bcl-2 or BAG-1 caused no significant difference among the transfectants in cell growth rates, either in vitro or in vivo. Taken together, these studies demonstrate that resistance to apoptosis is a crucial factor for development of peritoneal dissemination of human gastric cancer cells.
Abrogation of c-Raf expression induces apoptosis in tumor cells.
Lau QC. Brusselbach S. Muller R.
Institut fur Molekularbiologie und Tumorforschung, Philipps-Universitat Marburg, Germany.
Signal transduction pathways involving the c-Raf protein kinase are frequently activated in tumor cells. We have addressed the relevance of this activation by a loss-of-function approach. An anti-sense phosphorothioate oligonucleotide (ODN) specifically targeted against c-raf mRNA (Monia et al., 1996a) was used to block c-Raf protein expression in four different cell lines derived from lung, cervical, prostate and colon carcinomas. Concomitant with the abrogation of c-Raf expression we observed the occurrence of classical apoptotic markers, including chromatin condensation, inter-nucleosomal DNA cleavage, annexin V binding and cleavage of PARP, which was followed by cell death, affecting most of the cell population. This induction of apoptosis occurred independent of the p53 status of the cell. These findings demonstrate that c-Raf can protect tumor cells from undergoing programmed cell death, and suggest that the interference with c-Raf expression or function by ODNs or specific drugs could represent a powerful means for improving the efficacy of anti-cancer therapy.
The desmoplastic small round cell tumor t(11;22) translocation produces EWS/WT1 isoforms with differing oncogenic properties.
Kim J. Lee K. Pelletier J.
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Structural alterations of the Wilms' tumor locus (WT1) at 11p13 have been implicated in the etiology of two human cancers--Wilms' tumor (WT), a pediatric renal malignancy, and Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive cancer of the abdominal serosal lining with predilection for male adolescents. Germline mutations within the WT1 tumor suppressor gene predispose to WT and are associated with congenital malformations of the urogenital system, and somatic mutations are associated with initiation of transformation in WTs. In DSRCT, a recurrent translocation, t(11;22)(p13;q12), fuses the amino terminal domain of the EWS1 gene product to three of the four WT1 zinc fingers. Two EWS/WT1 isoforms are generated as a result of an alternative splicing event between zinc fingers III and IV, inserting or removing three amino acids (+/- KTS). We demonstrate that introduction of EWS/WT1(-KTS) into NIH3T3 cells causes their tumorigenic transformation as determined by: formation of transformed foci on a monolayer of cells; anchorage-independent growth; and tumor formation in nude mice. EWS/WT1(+KTS) showed no transforming potential in these assays. These results indicate the oncogenic effect of the t(11;22) translocation is mediated by the EWS/WT1(-KTS) isoform and that fusion of the EWS amino terminal domain to the WT1 DNA binding domain produces a chimeric product showing a gain of function.