Kapil N. Bhalla - US grants

The aim of this proposal is to evaluate the role of deoxycytidine (dCyd) as a growth protective agent for normal myeloid progenitor cells in antileukemic regimens consisting of dCyd analogs, or other pyrimidine antagonists, and high dose dCyd. This effort will attempt to uncover the biochemical mechanisms underlying a novel observation that supraphysiologic concentrations of this naturally occurring nucleoside selectively stimulates the in vitro growth of normal vs leukemic myeloid progenitor cells and preferentially protects the former from the lethal actions of certain pyrimidine antagonists, particularly 1-B- D-arabinofuranosylcytosine (Ara-C). Biochemical studies will be performed comparing the intracellular metabolism of high dose dCyd in freshly procured normal human bone marrow mononuclear cells and human leukemic myeloblasts. Specifically, we will study the intracellular degradation of dCyd and dCyd monophosphate (dCMP) and the accumulation of intracellular dCyd triphosphate (dCTP) pools (utilizing DNA polymerase assay). Similarly, both in normal and leukemic cells exposed to high dose dCyd/Ara-C regimens, we will carefully examine the accumulation of Ara- CTP; Ara-CTP to dCTP pool ratios (utilizing high pressure liquid chromatography methods); deamination of dCyd (dCMP) and Ara- C (Ara-CMP) by nucleoside and dCMP deaminase; incorporation of Ara-C into newly synthesized DNA; and molecular phenomenon such as the proportion of Ara-C residues at 3'-terminus of DNA strand to uncover possible explanations for the selective antileukemic effect of high dose dCyd/Ara-C regimens. In biologic studies, utilizing soft agar cloning methods, we will identify high dose dCyd and dCyd analog (e.g. 5-aza-Ara-C and 5- Aza-dCyd) dose relationship and schedules which exert lethal effects toward leukemic blast progenitors (L-CFU) while sparing normal myeloid elements (CFU-GM). The selective ability of dCyd to protect normal vs leukemic cells from other pyrimidine antagonists (e.g. thymidine and 3-deazauridine) will also be examined. Correlations with biochemical studies will be sought in order to test the hypothesis that impaired dCyd nucleotide formation or enhanced degradation in leukemic cells is responsible for diminished antagonism of pyrimidine antagonist mediated cytotoxicity. The ultimate objective of these studies is to generate in vitro data which will establish a rational basis for the design and implementation of clinical trails of high dose dCyd/dCyd analog, or high dose dCyd/pyrimidine antagonist, regimens in the treatment of acute nonlymphocytic leukemia.

DESCRIPTION: (Adapted from applicant's abstract) The aim of this application is to study the role of hematopoietic growth factors (HGFs) in improving the antileukemic selectivity of Ara-C, and to examine the biochemical and molecular mechanisms underlying this effect. These studies will expand on the applicant's previous findings that IL-3 and/or GM-CSF selectively enhance the metabolism and cytotoxicity of Ara-C against leukemic versus normal bone marrow progenitor cells.The effect of HGFs individually or in various combinations of early (stem cell growth factor, SCF), intermediate (IL-3 and GM-CSF) and lineage specific HGFs (G-CSF) will be examined on the cell cycle status (by flow cytometry) and the percentage of clonogenic S phase normal bone marrow progenitor cells (from healthy volunteers), AML blasts and established myeloid leukemia cell lines HL-60 and KG1. Following exposure to the HGFs in conjunction with Ara-C (1-100 uM) for different schedules and intervals, the applicant will compare the colony growth inhibition of normal (CFU-GM, CFU-GEMM and multipotent blast colonies) and leukemic (L-CFU) progenitor cells in semi-solid media. HGF combinations which in conjunction with Ara-C result in improved antileukemic selectivity of Ara-C will be examined for their effects on a variety of biochemical correlates of Ara-C cytotoxicity. These will include deoxycytidine kinase and deaminase activity, Ara-CTP accumulation, Ara-CTP/dCTP pool ratios (utilizing previously described HPLC methods) Ara-C DNA incorporation including the chain terminus position of Ara-C residues in normal and leukemic bone marrow elements. In addition, as a preliminary examination, the in vivo effects of GM-CSF on the intracellular metabolism of high dose Ara-C will be determined in the context of a laboratory companion study to a currently active cooperative group (CALGB) trial. To further explore the molecular mechanisms underlying the enhanced antileukemic activity of the combinations of HGFs and Ara-C, the applicant will examine his ability to produce programmed cell death (PCD) or apoptosis. Specifically, he will examine the ability of different doses and schedules of Ara-C with or without the HGFs to produce oligonucleosomal DNA fragmentation characteristic of PCD. These studies will also be performed in the presence of known modulators of protein kinase C (PKC) activity, phosphatase inhibitors and calcium ionophores. Ara-C mediated appearance of the characteristic ladder of DNA nucleosomal oligomers in agarose gel will be correlated with the altered expression on Northern blots of those genes which are known to regulate or are associated with the induction of PCD. These include bcl-2, c-jun, c-fos, c-myc and TGF-beta-1.These studies will explore the interaction of GHFs and Ara-C in activating PCD and correlate it with Ara-C DNA incorporation as well as the clonogenic survival of AML cells. The ultimate goal of these studies is to design rational antileukemic drug combinations of HGFs and Ara-C which will be based on an understanding of the biochemical and molecular mechanisms underlying the Ara-C mediated cytotoxicity.

DESCRIPTION: This application focuses on the use of paclitaxel and other chemotherapeutic agents in improving survival in patients with locally advanced (resectable) epithelial cancer of the esophagus (ECE). These investigators have recently found in a Phase I clinical and pharmacologic study that a relatively high dose of paclitaxel (250 mg/m2 over 3 hrs) in combination with cisplatin (CP) and 5-fluorouracil (FU) followed by G-CSF (TCFG regimen) is highly active in advanced ECE and produces a high rate of pathologic complete remission (pCR) in previously untreated, locally advanced resectable ECE. Ten patients with ECE were studied: six had advanced unresectable disease, all had a partial response (two-to-nine months); four had resectable ECE, all had clinical CR, two had pCR. Given the high response of the above trial, the applicants desire to conduct a formal Phase II study of the TCFG regimen as neo-adjuvant to surgery, and to study cellular molecular factors relevant to upstream (e.g., P-glycoprotein) and downstream (e.g., resistance to apoptosis) mechanisms of drug (paclitaxel) resistance in pretreatment ECE tumor specimens obtained from the patients in the trial. They have presented preliminary data obtained from human ECE cell lines that demonstrate that the overexpression of the antiapoptosis Bcl-2 and/or Bcl-XL proteins relative to the proapoptosis Bax protein represent a downstream mechanism of resistance to the cytotoxic effects and apoptosis caused by paclitaxel. They have also observed that loss of cell-cycle G1 checkpoint control represented by the loss of Rb or p16 protein or the overexpression of cyclin D1 and CDK4, as well as by the presence of a mutant p53 protein, are associated with an increased sensitivity of cultured ECE cells to paclitaxel-induced apoptosis. They have also measured, by immunoblot, the expression of Bcl-XL, Bcl-2, Bax, p16 INK4, cyclin D1 and P-glycoprotein in ECE tumor biopsy specimens, and have found that the expression of Bcl-XL, Bcl-2, Bax, and cyclin D1 was highly variable among the tumor samples. Given the above, the specific aims are as follows: 1. To perform a Phase II study of two cycles of TCFG regimen administered pre-operatively to determine the pathologic remission rates, duration of response and survival of patients with resectable ECE. Hypothesis: Addition of paclitaxel, an active agent against ECE that has non-overlapping mechanism of action and side effects with cisplatin and 5-FU would significantly improve the pCR rate in resectable ECE. 2. To determine the expression of Bcl-2, Bcl-XL, Bax and Bcl-XS in the pretreatment tumor samples of ECE, and to correlate these with the quality of remission in the esophagectomy specimen after pre-operative chemotherapy. Hypothesis: low Bcl-2 and/or Bcl-XL to Bax expression ratios in the pretreatment tumor samples correlate with the achievement of a pCR in ECE. 3. To examine the incidence of cyclin D1 and CDK4 overexpression and alterations in the Rb and p16 expressions in the pre-operative ECE tumor samples, in order to determine whether the loss of G1 checkpoint is a potential determinant of sensitivity of ECE to paclitaxel-based chemotherapy. Hypothesis: Loss of G1 checkpoint is common in ECE and is associated with pCR following paclitaxel-based chemotherapy of ECE.

DESCRIPTION: (Applicant's Abstract) Taxanes (taxol and taxotere) are highly active against human breast cancer. Taxane-induced microtubule bundling and CDK1 activation promotes prolonged mitotic arrest of cancer cells. This is associated with the pre-apoptotic mitochondrial permeability transition and release of cytochrome c, which in the cytosol binds to APAF-1 and triggers the cleavage and activity of caspase-9, followed by caspase-3, resulting in the morphologic and DNA fragmentation of apoptosis. Treatment with taxanes also causes the phosphorylation of Bcl-2 and Bcl-xL, which is associated with a rise in free Bax levels. Bcl-2 phosphorylation may negatively regulate its anti-apoptotic effect and mediate taxane-induced apoptosis. Bcl-2 or Bcl-xL blocks taxane-induced pre-apoptotic mitochondrial events and the activation of caspases, but does not affect microtubule bundling and mitotic arrest of cancer cells. Recently, apoptosis due to some chemotherapeutic drugs has been shown to involve Fas death receptor signaling. The recruitment of Fas signaling has not been examined in the context of taxane-induced apoptosis. Utilizing the relevant biologic sub-types of human breast cancer cells, this application would investigate the following molecular aspects of taxane activity: 1) the temporal and mechanistic relationship of the anti-microtubule and cell-cycle effects with taxane-induced pre-apoptotic mitochondrial perturbations, caspase activation and apoptosis; 2) the mechanistic role of Bcl-2 phosphorylation, the biologic activity of the 'loop' domain of Bcl-2 and Bcl-xL, as well as the impact of the levels of APAF-1, Bcl-2, and Bcl-xL relative to Bax, Bak, and Bad on taxane-induced apoptosis; 3) the role of Fas-signaling in taxane-induced apoptosis. Based on the observation that staurosporine (ST) enhances taxol-induced apoptosis, the mechanism of interaction as well as the optimum dosage and schedules of taxanes and UCN-01, a clinically relevant analogue of ST, would also be investigated against human breast cancer cells. These in vitro studies may define the potential molecular targets or elucidate mechanisms that could be manipulated to promote taxane-induced apoptosis of breast cancer cells.