Increased apoptosis and increased clonogenic survival of 12V-H-ras transformed rat fibroblasts in response to cisplatin

Mutationally activated Ras is involved in tumor progression and likely also in drug resistance. Using survival, viability and apoptosis assays, we have here compared the cisplatin sensitivities of FR3T3 rat fibroblasts and a 12V-H-ras transformed subline (Ras2:3). Around 24 h after cisplatin treatment Ras2:3 cells showed higher apoptosis levels and lower viability than FR3T3. This increased sensitivity correlated with weaker cisplatin-induced activation of Jun N-terminal kinase (JNK). In contrast to apoptosis assays, colony formation assays showed that Ras2:3 were more resistant to cisplatin than were FR3T3. This was partly due to the increased cisplatin sensitivity of FR3T3 seeded at low densities, as required in colony formation assays. In addition, Ras2:3 cisplatin survivors had a higher relative proliferative capacity. Cell cycle analyses showed that FR3T3 cells initially responded with a dose-dependent G2 arrest, while Ras2:3 accumulated in S-phase. Experiments with an anti-apoptotic mutant of MEKK1 suggested that the apoptotic response of Ras2:3 cells is not specific to the S-phase fraction. In summary, the cisplatin response of ras-transformed fibroblasts is distinct from that of parental cells, in that they show increased apoptosis, a different cell cycle response and increased post-treatment proliferative capacity. The results illustrate the need to carefully consider methods and protocols for in vitro studies on chemotherapy sensitivity.     

Radiation-induced apoptosis and its relationship to loss of clonogenic survival

Ionizing radiation can be an effective inducer of apoptosis and studies of many aspects of the pathways and mechanisms involved in this apoptosis induction have been published. This review stresses two aspects: the relationship between apoptosis and loss of clonogenic ability in irradiated cells and the time course for the appearance of apoptosis after radiation exposure. Although it was initially assumed that apoptosis occurred relatively quickly (within hours) after irradiation, evidence is presented and discussed here showing that apoptosis can occur at long times after irradiation (out to 20 days) in some cell types. This late, or delayed, apoptosis occurs after the cells have divided once or several times. The impact of delayed apoptosis on loss of clonogenicity after irradiation remains unclear. It seems likely that in some cell types, e.g., fibroblasts, the occurrence of late apoptosis is minimal and may have little impact on long term cell survival of the population, but in at least one instance, with a cell line of hematopoietic origin, it appears that late apoptosis can account for all the loss of clonogenicity in irradiated cells. The role of p53 in radiation-induced apoptosis is also discussed, with data presented showing that both p53-dependent and independent pathways for radiation-induced apoptosis exist, depending on the cell type.     

Automatic quantitation of cell colonies on petri dishes by computerized image analysis

Clonogenic assay is one of the most sensitive assays, widely used to evaluate the effects of antineoplastic agentsin vitro. A computer program was developed on an IBAS 2.0 Image Analysis System for automated quantiation of cell colonies and clone area on Petri dishes. The sensitivity of the clonogenic assay can be greatly increased by evaluating the mean area of the clones. The program gives an objective, accurate and fast evaluation of large samples. It is simple to use and offers a high degree of flexibility. Special algorithms and techniques have been implemented for good quantitation of both connected and well-separated colonies and to reduce the background noise and the general error rate. The principles and solutions presented are applicable to any other image analysis system.Abbreviations FBS fetal bovine serum - ATCC American Type Culture Collection - DDATHF 5,10-dideazatetrahydrofolic acid - PBS phosphate buffered saline     

Enhancement of rat ACT-1 tumor clonogenicity by xenogeneic mouse macrophages

Summary In vitro growth of rat atriocaval epithelial tumor cells (ACT-1) was enhanced by the inclusion of xenogeneic mouse adherent peritoneal exudate cells (PECs) in a two-layer soft agar system. A linear relationship was found between the number of cells plated and the number of colonies when ACT-1 tumor cells were plated at plating densities of between 1 and 5×10⁵ cell/60 mm plate (r=0.9,P<0.001). Inclusion of irradiated PECs in the bioassay for tumor stem cells resulted in a two and a half-fold increase in colony formation in three separate experiments (P<0.001). This work was supported by grants from the Cancer Research Trust, the University of Otago Cancer Research Fund and by the Medical Research Committee (Golden Kiwi).