Heat stress induced apoptosis in BC-8 cells derived from AK-5 tumor involves downregulation of Bcl-2 and generation of reactive oxygen species.

BC-8, a rat histiocytoma undergoes apoptosis after heat shock, which is due to lack of an effective heat shock response. Heat shock induced generation of free radicals, which in turn are involved in the induction of apoptotic death in BC-8 cells. Treatment of BC-8 cells with N-acetylcysteine partially inhibited the heat induced apoptosis. Introduction of Bcl-2 gene in these cells did not protect them from apoptotic death, whereas transfection with hsp-70 gene did render these cells resistant to heat induced apoptosis transiently. Heat shock also downregulated the expression of Bcl-2 and p53 in these cells. These observations suggested that the heat shock induced apoptosis was mediated through reactive oxygen species and controlled upstream of Bcl-2 check point.

Differential cytotoxic effects of Annona squamosa seed extracts on human tumour cell lines: role of reactive oxygen species and glutathione.

Annonaceous acetogenins are a new class of compounds that have been reported to have potent pesticidal, parasiticidal, anti-microbial, cell growth inhibitory activities. In this study, organic and aqueous extracts from the defatted seeds of Annona squamosa (custard apple) were tested on different human tumour cell lines for antitumoural activity. While organic and aqueous extracts induced apoptosis in MCF-7 and K-562 cells, they failed to do so in COLO-205 cells. Treatment of MCF-7 and K-562 cells with organic and aqueous extracts resulted in nuclear condensation, DNA fragmentation, induction of reactive oxygen species (ROS) generation and reduced intracellular glutathione levels. In addition downregulation of Bcl-2 and PS externalization by Annexin-V staining suggested induction of apoptosis in MCF-7 and K-562 cells by both the extracts through oxidative stress. On the contrary, COLO-205 cells showed only PS externalization but no change in ROS and glutathione levels. These observations suggest that the induction of apoptosis by A. squamosa extracts can be selective for certain types of cancerous cells.

Analysis of the p53 gene alterations in mouse embryo fibroblast cell line Balb 3T12 and its derivative 312.

We have analysed the status of the p53 gene in the mouse embryo fibroblast cell line Balb 3T12 (mso = 106) and its transformed clonal derivative 312 (mSO = 104) with an aim to determine whether there exists a correlation between increased tumorigenicity and clonal expansion of cells bearing a mutation in the p53 gene. While Southern hybridizations did not show any obvious changes in the p53 gene organization in 3T12 and 312 cells, sequencing the p53 cDNA revealed that 3T12 is mutated at the amino acid residue 233 (Tyr --+ Asp) whereas 312 is mutated at the residue 132 (Cys --+ Trp). Exploiting the altered RFLP pattern due to mutations, we identified that 3T12 contains p53 alleles that are different from the already identified mutant p53. On the basis of these observations, we conclude that 3T12 and 312 have evolved independently.

Mechanisms involved in natural killer cell mediated target cell death leading to spontaneous tumour regression.

We have studied the mechanisms involved in the spontaneous regression of a rat histiocytoma in syngeneic hosts and tumour cell death processes. In addition to the natural killer (NK) cells which act through antibody dependent cellular cytotoxicity (ADCC), TNF-α also participates in the induction of necrosis in tumours. We have shown that the tumour cell is killed by necrosis which is perforce mediated, and apoptosis leading to target cell DNA fragmentation. A prior activation of the effector cells is essential before it can kill the target cell, as naive effector cells are ineffective. Activation of effector cells is mediated by Thl type of cytokines in viro and in vivo. IFN-γ seems to play an important role in tumour regression as injection of antibodies to IFN- γ in vivo inhibited tumour rejection.

Suppression of tumourogenicity and overexpression of cell adhesion molecules in AK-5 cells transfected with wild-type p53 gene.

We have previously shown overexpression of p53 and rearrangement of the p53 gene in AK-5 tumour. In order to study the role of p53 in AK-5 tumourogenicity, we introduced wild-type p53 in AK-5 cells. We have shown suppression of tumourogenicity in AK-5 cells after transfixion with wt p53. In one of the transfected clones 3B4, there was complete loss of tumour forming ability. Clone 3B4 also showed cellular aggregation which correlated well with the higher expression of cell adhesion molecules like fibronectin and hyaluronectin. These observations demonstrate tumour suppression in AK-5 after introduction of wt p53.