Govindaswamy Shanmugam

Promoter hypermethylation profile of tumor‐associated genes p16, p15, hMLH1, MGMT and E‐cadherin in oral squamous cell carcinoma

Aberrant promoter hypermethylation of tumor‐associated genes leading to their inactivation is a common event in many cancer types. Using a sensitive restriction‐multiplex PCR method, we studied the promoter hypermethylation profile of the p16, p15, hMLH1, MGMT and E‐cad genes in oral squamous cell carcinoma (OSCC) of Indians. We analyzed a total of 51 samples for the p15 tumor‐suppressor gene and 99 samples for each of the remaining genes. Our studies indicate an incidence of promoter hypermethylation of 23% each for p16 and p15, 8% for hMLH1, 41% for MGMT and 35% for E‐cad. We observed aberrant hypermethylation of the promoter region of at least 1 of these genes in 74.5% of cases (n = 51) for which all the 5 genes were studied. Abnormal methylation was detected in tumors irrespective of stage and location in the oral cavity, whereas no abnormal methylation was detectable in normal oral squamous tissues obtained from 25 OSCC patients. Detection of aberrant hypermethylation patterns of cancer‐associated genes listed above is therefore suitable for diagnosis of OSCC in individuals at high risk for this disease.

p53 gene mutations in oral carcinomas from India

In this study, we analyzed 53 oral squamous‐cell carcinomas among Indians for the presence of alterations in the tumor‐suppressor gene p53 by PCR‐SSCP and sequencing methods. Our results showed that 21% (II/53) of oral carcinomas analyzed carried mutations within the exons 5–8 of the p53 gene. We have identified II single‐base pair substitutions consisting of 10 mis‐sense mutations and one at the splice acceptor site, and one deletion mutation involving 4 consecutive bases. The majority of the base substitutions were transitions (5 TA to CG and 5 GC to AT), while only one transversion (TA to GC) was observed. Probable hot‐spots for the mutation induction were identified at codons 149 and 274, which have not been observed before in head‐and‐neck cancers. The mutational spectrum might have originated from base alkylations at guanine and thymine residues, caused by some alkylating agents. The present results are thus consistent with the involvement of tobacco‐related nitrosoamines in the etiology of oral squamous‐cell carcinoma.

Mutational analysis of the candidate tumor suppressor gene ING1 in Indian oral squamous cell carcinoma

ING1, a recently identified candidate tumor suppressor gene, involved in the p53 signaling pathway is mapped at chromosome 13q34. Since loss of heterozygosity at 13q34 has been reported in squamous cell carcinoma of head and neck, we screened for mutations in ING1 by polymerase chain reaction-single strand conformation polymorphism in 71 oral squamous cell carcinomas (OSCC) from India, 15 of which were known to harbor p53 mutations. A single polymorphism (G to A) was detected in 14 (19.7%) of the tumors analyzed. No mutation was observed in any of the 71 OSCCs analyzed. These results suggest that ING1 is not a target for mutational inactivation in OSCC of Indians.

Genotoxicity of the herbicide butachlor in cultured human lymphocytes

Butachlor, a pre-emergence herbicide was investigated for its ability to induce sister chromatid exchanges (SCE) and chromosome aberrations (CA) in cultured human peripheral blood lymphocytes. Mitogen-stimulated lymphocytes were treated with three different concentrations (5, 10 and 20 micrograms/ml) of butachlor for 24, 48 and 72 h. Our results indicate a dose-dependent increase in the frequency of chromosomal aberrations at 24, 48 and 72 h of treatment with butachlor. No SCE was promoted by butachlor.

Cleistanthin A causes DNA strand breaks and induces apoptosis in cultured cells

Cleistanthin A is a novel anticancer agent isolated from Cleistanthus collinus (Rox B). It caused chromatid aberrations in a dose dependent manner. However, the concentrations that induced the aberrations, neither affected viability nor induced DNA strand breaks. Only at higher concentrations and after long exposure, DNA strand breaks were observed. Cleistanthin A induced apoptosis in Chinese hamster ovary (CHO) cells, in cervical carcinoma (Si Ha) cells and in a p53 deficient cell line K562. Cleistanthin A-induced cell death was low in bcl-2 transfected cells. Cleistanthin A inhibited the incorporation of [3H]thymidine into DNA; however, it did not affect the transport of [3H]thymidine into these cells. These studies indicate that the cytotoxic effects of cleistanthin A are mediated by the inhibition of DNA synthesis, induction of DNA damage and apoptosis.

Genotoxicity of the herbicide fluchloralin on human lymphocytes in vitro: chromosomal aberration and micronucleus tests.

Cultured human lymphocytes were exposed to three different concentrations (2.5, 5.0 and 10.0 micrograms/ml) of fluchloralin for 24 and 48 h to assess chromosomal aberrations. A significant dose-dependent increase of chromatid type aberration was observed in these cells. Multiple aberrations (MA) were scored at all concentrations after 48 h treatment. To support these results we did micronucleus (MN) test using cytochalasin B to block cytokinesis. At lower concentrations (2.5 to 10.0 micrograms/ml) the frequency of MN induction was not significantly different. Higher concentrations of fluchloralin (20, 40 and 50 micrograms/ml) resulted in a significant dose dependent increase in number of micronucleated cells. This is the first report on genotoxic effects of fluchloralin in human cells.

Expression of bcl-2 oncoprotein in Indian oral squamous cell carcinomas

Thirty-nine oral squamous cell carcinomas were assessed for bcl-2 protein expression by immunostaining of tumour sections. Twenty-three per cent of these tumours showed strong nuclear staining for bcl-2 protein. Tumours of the cheek and tongue together accounted for 77% of overexpression of this protein. When bcl-2 expression was compared with p53 expression, they were found to be non-overlapping. These results suggest that overexpression of either of these genes may substitute each other in the development of oral carcinomas of Indians.

Selective deletion of p14(ARF) exon 1β of the INK4a locus in oral squamous cell carcinomas of Indians

The tumor suppressor gene - p16 INK4/CDKN2/MTS1 and its alternate splice product p14 (ARF), constitute the INK4a locus. We have examined the integrity of exon 1beta of p14(ARF) gene of oral squamous cell carcinomas (n=58) in untreated Indian patients. No mutations were detected in this region by PCR-SSCP analysis of the tumor DNAs. Further, PCR-based analysis revealed homozygous deletions of exon 1beta in 14 of the 58 tumors; these results were confirmed by hybridization of tumor DNAs with exon 1beta specific probe. The deletions were limited to the exon 1beta while the exons coding p16/INK4 were not affected. Except in two cases these deletions were mutually exclusive to the p53 inactivating mutations. These observations suggest an alternate mechanism of loss of p14(ARF) in the genesis of oral squamous cell carcinomas.

Cleistanthin B causes G1 arrest and induces apoptosis in mammalian cells.

Cleistanthin B is a potential anticancer agent isolated from the tropical plant Cleistanthus collinus. We have previously shown that cleistanthin B is clastogenic and induces micronuclei formation and chromosomal aberrations. We now show that this compound inhibits DNA synthesis in Chinese hamster ovary (CHO) cells and induces apoptosis in cervical carcinoma (SiHa) cells. Flow cytometric analysis of cleistanthin treated CHO cells revealed that they were blocked in G1. Cervical carcinoma (SiHa) cells exposed to cleistanthin B shrank, rounded up and had condensed chromatin and fragmented nuclei. DNA isolated from cleistanthin treated cells exhibited the characteristic apoptotic ladder when electrophoresed in agarose gels. These results were confirmed by flow cytometry. Etoposide, a structurally similar compound also induced apoptosis in these cells although with a difference. Etoposide induced apoptosis after permitting cells to enter into S phase, while cleistanthin B stopped entry of cells into S phase and subsequently drove them to apoptosis.

Cleistanthin A, a diphyllin glycoside from Cleistanthus collinus, is cytotoxic to PHA-stimulated (proliferating) human lymphocytes

An ideal anticancer drug would be one that preferentially kills tumor cells with the least toxicity to normal cells. Cleistanthin A, a diphyllin glycoside of the tropical plant Cleistanthus collinus, was found to possess cytotoxic and tumor regressing properties. To find out whether this compound acts selectively on proliferating cells it was tested against quiescent and proliferating human lymphocytes. Mitogen‐stimulated and unstimulated human lymphocytes were treated with cleistanthin A. A cytotoxicity assay using MTT was used to assess the viability of the cells. Percentage viability of the unstimulated and treated cells were normalized to that of the untreated and unstimulated cells and percentage viability of stimulated and treated cells were normalized to that of stimulated and untreated cells. Quiescent lymphocytes were refractory to the action of cleistanthin A. Only proliferating cells were killed. Cell death was proportional to the percentage of cells in the proliferating stage and was also dose‐dependent. Quiescent lymphocytes pretreated with cleistanthin A had the ability to proliferate upon subsequent stimulation with PHA. These results indicate that cleistanthin A does not affect the viability of quiescent cells. Also, it did not affect the proliferating potential of quiescent cells. However, this compound drastically affected proliferating cells by reducing their viability to 10–20%. Our results therefore indicate that the antiproliferative property of cleistanthin A could be used in regimens for treating tumors with extensive proliferative potencies. Drug Dev. Res. 51:187–190, 2000.