| Protein kinase C (PKC) signaling events influence apoptosis and the cytotoxicity of cytosine arabinoside is affected by PKC signaling in leukemic cells. This dissertation presents findings that extend these observations to include another deoxycytidine analog, gemcitabine (dFdC), in a solid tumor model. DFdC induced apoptosis in BG-1 human ovarian cancer cells based on biochemical and morphological analyses. Scanning and transmission electron microscopy of BG-1 cells showed morphologic changes associated with apoptosis in response to dFdC: membrane blebbing, the formation of apoptotic bodies and chromatin condensation. Time-lapse video microscopy of BG-1 cells exposed to dFdC indicated that the kinetics of dFdC-induced apoptosis were protracted; the apoptotic events were asynchronous and cumulative. The majority of cell deaths occurred within 24-48 hours of dFdC treatment. Annexin V-FITC positive staining of phosphatidylserine, translocated to the outer surface of the BG-1 cell plasma membranes, was evident, but unremarkable. Caspase 3 activity was not evident before 12 hours of dFdC treatment, but once elevated, remained elevated throughout 48 hours of drug exposure. Exposure to dFdC induced high molecular weight (50 kbp) DNA fragmentation in BG-1 cells, but internucleosomal (200 bp) fragmentation was not detected. Furthermore, dFdC activated PKC in BG-1 cells based on increases observed, following dFdC treatment, in PKC enzyme activity, phosphorylation of the endogenous PKC substrate myristoylated alanine-rich C kinase substrate, and cytosol to membrane translocation of PKC isozymes. Moreover, clonogenicity studies with dFdC in combination with PKC modulating agents demonstrated that dFdC cytotoxicity was influenced by PKC signaling events. DFdC cytotoxicity was increased synergistically when drug treatment was, followed or preceded, by chronic (24 hour) exposures to TPA. These observations prompted further investigation of PKC signaling events linked to TPA- and dFdC-induced cytotoxicity in BG-1 cells. Since PKC isozymes are differentially expressed in various cell types, it was determined that BG-1 cells express the {dollar}alpha, beta, delta, varepsilon,{dollar} and {dollar}zeta{dollar} isoforms of PKC. The PKC mechanism(s) responsible for the synergism of TPA and dFdC were then explored. Chronic treatment of BG-1 cells with TPA: (1) down-regulated PKCs {dollar}alpha{dollar} and {dollar}delta,{dollar} but had no effect on PKC{dollar}varepsilon,{dollar} (2) did not affect cell cycle distribution into S phase, (3) increased ERK/MAPK signaling, and (4) increased intracellular alkaline phosphatase, a biochemical marker for cellular differentiation. Thus, chronic exposure to TPA enhanced dFdC cytotoxicity by inducing differentiation pathways. It is important to recognize that modulating signal transduction pathways can improve chemotherapeutic drug response. However, different signaling pathways influence drug cytotoxicity and modulating these pathways can lead to drug synergism or antagonism. Therefore, understanding the signaling mechanisms that influence drug response is essential. |
