Expression of cell death regulatory genes and limited apoptosis induction in avian blastodermal cells.

Apoptosis is a well-established cellular mechanism for selective cell deletion during development. However, little is known about the expression of an apoptotic pathway and its role in determining the relative sensitivity of the early, pre-gastrula, avian embryo to stress-induced cell death. We examined the sensitivity of avian blastodermal cells to engage in apoptosis upon exposure to etoposide, a topoisomerase II-inhibitor that rapidly and efficiently induces apoptosis in many cell types. We found that while the blastodermal cells are capable of engaging in apoptosis, they are highly resistant to such induction with respect to both concentration of drug required and length of exposure, even when compared to a tumor cell line with a known multi-drug resistant phenotype. Additionally, we assessed the expression of several candidate regulatory genes in blastodiscs from infertile eggs (i.e., maternal RNA transcripts), blastodermal cells immediately following oviposition, and various stages of early development up to gastrulation. This analysis revealed that several genes whose products have anti-apoptotic activity, including bcl-2, bcl-xL, hsp70, grp78 and the glutathione S-transferases, are expressed as early as the stage 1 embryo in the newly oviposited egg. These transcripts are also found in the infertile blastodisc, suggesting a role for maternally derived transcripts in the protection of the oocyte and zygote. Significantly, constitutive levels of hsp70 mRNA exceeded those of the other anti-apoptotic genes in the blastodermal cells. These results contribute to an emerging picture of stress resistance at the earliest stages of avian embryo development which involves multiple anti-apoptotic genes that act at different regulatory points in the apoptotic cascade.

Contribution of gene-specific lesions, DNA-replication-associated damage, and subsequent transcriptional inhibition in topoisomerase inhibitor-mediated apoptosis in lymphoma cells.

Lymphoid lineage tumor cells differ widely in their relative sensitivity or resistance to the induction of apoptosis by a variety of chemotherapeutic drugs. We used a model system of virally transformed B- and T-lymphoma cell lines to show that avian T-lymphoma cells are highly resistant, whereas B-lymphoma cells are highly sensitive, to the induction of apoptosis by a wide spectrum of chemotherapeutic drugs that induce different types of lesions in DNA. Among the various drugs examined, the topoisomerase inhibitors, camptothecin, actinomycin D, and etoposide, were the most potent inducers of apoptosis. Examination of the relative contribution of DNA replication and transcriptional inhibition to the differential induction of apoptosis by the topoisomerase inhibitors revealed that the signals initiating the apoptotic response vary, even among compounds with similar cellular targets. Specifically, DNA replication plays a major role in the induction of camptothecin-induced apoptosis, and a lesser role in the induction of apoptosis by etoposide. In contrast, DNA replication is not involved in the induction of apoptosis by actinomycin D. Transcriptional inhibition may provide the major cellular signal for apoptosis induction by this compound. In addition, we determined that the extent of topoisomerase I-cleavable complex inhibition is similar even in genes that are transcribed at different levels and by different RNA polymerases. An overexpressed c-myc gene is no more vulnerable to topoisomerase inhibition than its normally expressed counterpart. In contrast, even under conditions yielding similar amounts of topoisomerase inhibition, rRNA genes are more sensitive to transcriptional inhibition than are the c-myc genes.