PubMed Health⌕ Search

Biomedical subjects

S Markowitz

Publications and source records attributed to S Markowitz.

At least 37 records · Page 2Linked to original sources
PubMed1998

[Investigation of the genetic instability of tumor cells by transfection of RER+ cell lines with exogenous microsatellite sequence].

OBJECTIVE: Frequent alterations of microsatellite sequence of cancer cells were found recently in a substantial fraction of human cancers including hereditary non-polyposis colon cancer. This paper aimed to investigate the genetic instability of tumor cells by using microsatellite instability(MI) as the marker in vitro. METHODS: Two RER+ cell lines (replication error phenotype), RKO and HCT116 and one RER- cell line, sw480, were used as the hosts for transfection with an episomal plasmid, pCMV-CAR, containing an exogenous (CA)14 repeat which was inserted within the coding sequence of lacZ reporter gene and thus made lacZ misreading. The transfectant clones were selected and established by hygromicin. Expression and production of lacZ reporter gene of restored reading frame were detected with X-gal staining assay. RESULTS: After hygromicin selection, stable pCMV-CAR transfectant clones were established. It was shown that mutation of deletion of insertion within(CA)14 occurred in the transfectant RER+ cells but not in the RER-cells. The mutation restored normal reading frame of lacZ gene, and resulted in expression and production of bio-active beta galactosidase which was detected with X-gal staining. This feature of the transfectant clones was maintained during culture passages. CONCLUSION: The alterations of the exogenous(CA)14 repeat in the transfectant RER+ clones revealed genetic instability and complicated mutation status of cancer cells. It is suggested that the exogenous (CA)14 in transfectant clones could be a useful target sequence for monitoring the effects of environmental agents on MI of human cancer cells.

Cell Line↗
PubMed1996

Mismatch repair mutations override alkyltransferase in conferring resistance to temozolomide but not to 1,3-bis(2-chloroethyl)nitrosourea.

Cells with the mutator phenotype are tolerant to methylating damage from N-methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanine, exhibit replication repair errors, and have recently been found to be mutant in mismatch repair (MMR). However, resistance of cell lines with these defects to clinically used chemotherapeutic agents and the relationship of this resistance to expression of O6-alkylguanine-DNA alkyltransferase (AGT), which repairs DNA damage caused by methylating agents, has not been demonstrated. We compared resistance to the methylating agent temozolomide (TMZ) and to the chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), with and without AGT inhibition by 06-bG in several colorectal carcinoma cell lines. Two cell lines had known microsatellite instability (replication repair error-positive) and high levels of AGT, as well as a mutation in one of two MMR genes, hMLH1 (HCT116) or GTBP (HCT15). Cell line SW480 had wild-type MMR genes and high AGT, and HCT116+Ch3 has previously been transduced with chromosome 3 (carrying wild-type hMLH1) and thus has a "corrected" MMR phenotype. SW480 exhibited the expected sensitivity to TMZ and BCNU and marked potentiation of cytotoxicity by O6-bG. In contrast, HCT15 and HCT116 were markedly resistant to TMZ and were not sensitized by O6-bG-mediated inhibition of AGT, whereas the sensitivity pattern in HCT116+Ch3 cells was similar to that in SW480. All cell lines were sensitized to BCNU by O6-bG. Thus, tumor cells with defects in MMR appear particularly resistant to methylating agents in a manner that overrides dependence on AGT and its inhibition by O6-bG. However, these cells use AGT for resistance to chloroethylating agents, providing an alternative strategy for alkylating agent therapy.

Antineoplastic Agents, Alkylating↗
PubMed1996

Expression of hyaluronidase by tumor cells induces angiogenesis in vivo.

Hyaluronic acid is a proteoglycan present in the extracellular matrix and is important for the maintenance of tissue architecture. Depolymerization of hyaluronic acid may facilitate tumor invasion. In addition, oligosaccharides of hyaluronic acid have been reported to induce angiogenesis. We report here that a hyaluronidase similar to the one on human sperm is expressed by metastatic human melanoma, colon carcinoma, and glioblastoma cell lines and by tumor biopsies from patients with colorectal carcinomas, but not by tissues from normal colon. Moreover, angiogenesis is induced by hyaluronidase+ tumor cells but not hyaluronidase- tumor cells and can be blocked by an inhibitor of hyaluronidase. Tumor cells thus use hyaluronidase as one of the "molecular saboteurs" to depolymerize hyaluronic acid to facilitate invasion. As a consequence, breakdown products of hyaluronic acid can further promote tumor establishment by inducing angiogenesis. Hyaluronidase on tumor cells may provide a target for anti-neoplastic drugs.

Animals↗
PubMed1996

Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers.

Chromosome deletions are the most common genetic events observed in cancer. These deletions are generally thought to reflect the existence of a tumour suppressor gene within the lost region. However, when the lost region does not precisely coincide with a hereditary cancer locus, identification of the putative tumour suppressor gene (target of the deletion) can be problematic. For example, previous studies have demonstrated that chromosome 18q is lost in over 60% of colorectal as well as in other cancers, but the lost region could not be precisely determined. Here we present a rigorous strategy for mapping and evaluating allelic deletions in sporadic tumours, and apply it to the evaluation of chromosome 18 in colorectal cancers. Using this approach, we define a minimally lost region (MLR) on chromosome 18q21, which contains at least two candidate tumour suppressor genes, DPC4 and DCC. The analysis further suggested genetic heterogeneity, with DPC4 the deletion target in up to a third of the cases and DCC or a neighbouring gene the target in the remaining tumours.

Alleles↗
PubMed1996

Clinical analysis of the utility of repeat computed tomographic scan before discharge in blunt hepatic injury.

OBJECTIVES: Nonoperative management of hemodynamically stable blunt hepatic injury has emerged as an acceptable and safe treatment. Surveillance of this population's injuries is costly. As a prelude to establishing practice guidelines, the utility of repeat computed tomographic (CT) scans was investigated. METHODS: A retrospective study was conducted on 243 hepatic injuries. The CT scans of 95 patients managed nonoperatively who did not have ongoing transfusion requirements were reviewed and graded according to the American Association for the Surgery of Trauma (AAST) hepatic injury scale. Patients were grouped according to injury grade, assigned to two subgroups (patients with one CT scan versus more than one CT scan) and compared with respect to several physiologic and clinical variables. RESULTS: Statistical analysis revealed no significant difference between subgroups with the same grade of injury. No significant difference was demonstrated between subgroups' length of stay. CONCLUSIONS: No patients failed nonoperative treatment or succumbed to their injuries. Findings on repeat CT scan have not altered the decision to discharge the clinically stable patient having suffered a grade III or lower liver injury.

Humans↗
PubMed1996

Spontaneous apoptosis in human colon tumor cell lines and the relation of wt p53 to apoptosis.

OBJECTIVE: To examine spontaneous apoptosis of cultured human colon tumor cell lines in vitro and to investigate the role of wild type (wt) p53 in regulation of apoptosis induced by DNA-damaging treatment. METHODS: A model system of human tumor progression involving three cell lines was used in this study for examination of apoptosis. They were originally established from human colon villous adenoma, including an early passage of non-tumorigenic cell line, V235E; a late passage of weakly tumorigenic cell line, V235L; and a spontaneous progressing highly tumorigenic cell line. V411. All of them maintain wt p53 expression. For identification of apoptosis, two tests were performed: 1. morphology study using acridine orange (AO)/ethidium bromide (EB) stainning by fluorescence microscopy; 2. DNA electrophoresis on agarose gel. P53 and WAF-1 (a downstream gene of p53) expressions were analysed at mRNA level using Northern blot technique. Apoptotic index of cell lines examined was measured by DNA fluorescence assay. RESULTS: Spontaneous apoptosis was demonstrated in cell lines of all stages of progression by both morphology and DNA agarose gel electrophoresis. Apoptosis was further induced in V411 after treatment of cells with 137Cs gamma-irradiation and accompanied by increases in p53 and WAF-1 expression. In contrast, a mutant p53 bearing human colon cancer cell line, sw480, lacked spontaneous apoptosis, and upon irradiation neither induction of apoptosis nor increase expression of p53 and WAF-1 were seen. CONCLUSIONS: Apoptosis can be maintained in some human tumor cell lines despite transformation and carcinogenesis. Wt p53 and WAF-1 products are two of the potential mediators which effect apoptosis. Additionally, since apoptosis was enhanced by irradiation in V411, but not in sw480, it suggests that wt p53 cancer cells are more sensitive to DNA-damaging treatment than mutant p53 cancer cells. These finding may have implications for cancer therapy.

Adenoma, Villous↗
PubMed1995

Absence of secretory phospholipase A2 gene alterations in human colorectal cancer.

A potent modifying locus of intestinal tumorigenesis in the mouse was recently identified as secretory phospholipase A2 (sPLA2). The human homologue of sPLA2 maps to chromosome 1p35, a region frequently lost in human tumors. To evaluate the possibility that sPLA2 was a tumor suppressor gene that was the target of the 1p loss events, we identified polymorphisms within the human sPLA2 gene. Using these polymorphisms, 31% of 16 colorectal carcinomas were found to lose a sPLA2 allele. However, sequence analysis of the complete coding region of sPLA2 revealed no somatic mutations in the remaining allele of those tumors with allelic loss, nor in 18 additional colorectal cancers. Thus, sPLA2 is within the chromosomal region often lost during colorectal tumorigenesis, but mutations of this gene do not appear to play a major role in colorectal cancer development, and sPLA2 is unlikely to be the 1p35 tumor suppressor.

Animals↗
PubMed1995

Both transforming growth factor-beta and substrate release are inducers of apoptosis in a human colon adenoma cell line.

VACO-330, a nontransformed cell line established from a human colon adenoma, undergoes spontaneous apoptosis and shedding of cells into the culture medium. Shed cells were shown to be apoptotic, both by nuclear morphology and by generation of a typical "laddered" pattern of degraded DNA. Quantitation of DNA released into the medium, compared with the amount retained on the plate, demonstrated that 6.2 +/- 1.1% of the total cell mass underwent apoptotic death daily. The addition of transforming growth factor beta (20 ng/ml) accelerated this spontaneous apoptotic rate 3.2-fold. Moreover, apoptosis could be rapidly induced in up to 45% of the VACO-330 cells by using brief exposure to a calcium chelating medium to release the cells from the substratum. We suggest that transforming growth factor beta is a likely physiological regulator of apoptosis during maturation of the colonic epithelial cells. We additionally suggest the existence of an alternate pathway, which at the time of shedding from the crypt induces apoptosis in colonic epithelial cells that have escaped earlier apoptotic signals.

Adenoma↗
PubMed1995

Demonstration that mutation of the type II transforming growth factor beta receptor inactivates its tumor suppressor activity in replication error-positive colon carcinoma cells.

Escape from negative growth regulation by transforming growth factor beta (TGF-beta) as a result of the loss of TGF-beta type II receptor (RII) expression has been found to be associated with the replication error (RER) colorectal cancer genotype, which is characteristic of hereditary nonpolyposis colorectal cancers. The RER-positive HCT 116 colon carcinoma cell line was examined for RII mutations. A 1-base deletion was found within a sequence of 10 repeating adenines (nucleotides 709-718), which resulted in a frameshift mutation. Although it is reasonable to predict that the loss of RII function would be an important determinant of malignancy, the large number of potential mutations in cells of this phenotype raises the possibility that an RII mutation may not be a key event in the tumorigenic phenotype of these cells. One way to test directly the importance of RII mutations in determining the malignant phenotype would be to restore its expression. If restoration of expression leads to diminished tumorigenicity, it would indicate that RII mutation is an important determinant of malignancy in the RER phenotype. To determine whether restoration of RII would lead to reversal of malignancy in RER colon cancers, an RII expression vector was transfected into the HCT 116 cell line. RII stable clones showed mRNA and protein expression of transfected RII. The fibronectin mRNA level was increased by exogenous TGF-beta 1 treatment in a dose-dependent manner in RII-positive clones, whereas the control cells remained insensitive. The RII transfectants showed reduced clonogenicity in both monolayer culture and soft agarose. They were growth arrested at a lower saturation density than control cells. TGF-beta 1-neutralizing antibody stimulated the proliferation of RII-transfected but not control cells, indicating that the alterations in the growth parameters of the transfected cells were due to the acquisition of autocrine-negative activity. Tumorigenicity in athymic mice was reduced and delayed in RII transfectants. These results indicate that reconstitution of TGF-beta autocrine activity by reexpression of RII can reverse malignancy in RER colon cancers, thus verifying that the malignancy of hereditary nonpolyposis colorectal cancer can be directly associated with the loss of RII expression.

Animals↗
PubMed1995

Mutations of GTBP in genetically unstable cells.

The molecular defects responsible for tumor cell hypermutability in humans have not yet been fully identified. Here the gene encoding a G/T mismatch-binding protein (GTBP) was localized to within 1 megabase of the related hMSH2 gene on chromosome 2 and was found to be inactivated in three hypermutable cell lines. Unlike cells defective in other mismatch repair genes, which display widespread alterations in mononucleotide, dinucleotide, and other simple repeated sequences, the GTBP-deficient cells showed alterations primarily in mononucleotide tracts. These results suggest that GTBP is important for maintaining the integrity of the human genome and document molecular defects accounting for variation in mutator phenotype.

Base Sequence↗
PubMed1995

Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability.

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of epithelial cell growth. Human colon cancer cell lines with high rates of microsatellite instability were found to harbor mutations in the type II TGF-beta receptor (RII) gene. Eight such examples, due to three different mutations, were identified. The mutations were clustered within small repeated sequences in the RII gene, were accompanied by the absence of cell surface RII receptors, and were usually associated with small amounts of RII transcript. RII mutation, by inducing the escape of cells from TGF-beta-mediated growth control, links DNA repair defects with a specific pathway of tumor progression.

Amino Acid Sequence↗
PubMed1995

Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability.

Microsatellite instability has been observed in both sporadic and hereditary forms of colorectal cancer. In the hereditary form, this instability is generally due to germline mutations in mismatch repair (MMR) genes. However, only one in ten patients with sporadic tumours exhibiting microsatellite instability had a detectable germline mutation. Moreover, only three of seven sporadic tumour cell lines with microsatellite instability had mutations in a MMR gene, and these mutations could occur somatically. These results demonstrate that tumours can acquire somatic mutations that presumably do not directly affect cell growth but result only in genetic instability. They also suggest that many sporadic tumours with microsatellite instability have alterations in genes other than the four now known to participate in MMR.

Adult↗
PubMed1995

Mutant K-ras oncogenes in colon cancers Do not predict Patient's chemotherapy response or survival

One half of human colon cancers bear mutant c-K-ras oncogenes. Mutant K-ras oncogenes are associated with shortened survival in non-small cell lung cancers, and, in cell line models, with resistance to cis-platinum and to ionizing radiation. This study examines whether mutant K-ras alleles in colon cancer alter patients' response to chemotherapy or survival. We studied 37 patients who received chemotherapy with 5-fluorouracil and leucovorin, Exon 1 of the c-K-ras gene was PCR amplified from DNA extracted from paraffin-embedded tumor blocks. The presence of mutant or wild-type c-K-ras alleles was determined by dideoxy sequencing of the PCR-amplified c-K-ras DNA. c-K-ras mutations at codons 12 or 13 were present in 19 and absent in 18 cases. Responses to chemotherapy were equally likely in patients with either wild-type or mutant c-K-ras, occurring in 28% of patients with wild-type ras and 32% of patients with mutant ras (P = 0.8). Survival was also indistinguishable among both groups. Median survival from diagnosis was 35 months for ras wild-type patients and 31 months for ras mutant patients (P = 0.96). Median survival from starting chemotherapy was 14 months for ras wild-type patients and 17 months for ras mutant patients (P = 0.26). Patients with colon cancers bearing either wild-type or mutant c-K-ras alleles are indistinguishable in overall survival and are equally likely to respond to 5-fluorouracil-based chemotherapy.

Journal Article↗
PubMed1994

Deletion of p16 and p15 genes in brain tumors.

We have used molecular genetic methods to examine the status of cell cycle-inhibitory genes in human brain tumors. We found that p16 and a neighboring gene, p15, were often homozygously deleted in glioblastoma multiformes but not in medulloblastomas or ependymomas. The deletions occurred in both primary tumors and their derived xenografts, but no intragenic mutations in either of the two genes were found. The p15 gene was expressed in a more widespread pattern in normal tissues than p16, but the products of both genes had similar capacities to bind to cyclin D-dependent kinases 4 and 6. These data suggest that the target of deletion in glioblastoma multiforme includes both p15 and p16 genes. The reason that homozygous deletions, rather than intragenic mutations, are so common in these tumors may be that deletion is a more efficient mechanism for simultaneous inactivation of both genes.

Animals↗
PubMed1994

hMSH2 mutations in hereditary nonpolyposis colorectal cancer kindreds.

It has recently been shown that hereditary nonpolyposis colorectal cancer (HNPCC) is caused by hereditable defects in DNA mismatch repair genes. However, the fraction of HNPCC due to defects in any one repair gene and the nature of these mutations are not known. We analyzed 29 HNPCC kindreds for mutations in the prototype DNA mismatch repair gene hMSH2 by a combination of linkage analysis, polymerase chain reaction-based screening, and sequencing of the coding region. The complete intron/exon structure of the gene was ascertained to facilitate this analysis. The results suggest that at least 40% of classic HNPCC kindreds are associated with germline mutations in hMSH2 and that most of these mutations produce drastic alterations in the predicted protein product.

Amino Acid Sequence↗