The proposal's central hypothesis is that metabolism of polycyclic aromatic hydrocarbons by CYP1B1 in bone marrow stromal cells plays an integral role in progenitor B cell apoptosis. Scientific Merit:
Specific Aim 1. Confirm whether the bone marrow stromal cells (BMS2) metabolize DMBA primarily using CYP1B1. The preliminary data have demonstrated that the bone marrow stromal cell line, BMS2, metabolize DMBA in a metabolic profile consistent with CYP1B1 activity. Moreover, with respect to TCDD induction of CYP1B1, this cell line re-capitulates primary bone marrow stem cells (PBMS). Finally, the CYP1B1 activity is readily detected, constituitively, in both BMS2 and PBMS, thus suggesting that induction of CYP1B1 is not necessary for DMBA metabolism. The strengths of this aim is that the model system has the appropriate metabolic activity and inducibility. Further experiments are proposed to address the possible concern that CYP1A1 (although not immuno-detectable) may perform this activity, i.e., DMBA metabolism. The candidate will also determine if the possible apoptotic targets of DMBA metabolites, pro-B cells, have this metabolic activity-although previous studies suggest that this is unlikely. An additional strength is that advisor, Dr. Jefcoate, has assays and reagents available to evaluate DMBA metabolism. Further experiments using the combination of specific and non-specific P450 inhibitors and anti-sera should allow the applicant to determine with some degree of certainty whether CYP1B1 is involved in the metabolism of DMBA and whether the pro-B-cells have this capability.
Aim 2 assesses whether DMBA metabolism by BMS2 cells is required for progenitor B cell apoptosis.
This aim i s developed to extend some recent findings suggesting that DMBA apoptosis of pre-B cells is dependent upon bone marrow stromal cells. In the absence of these cells, DMBA did not cause apoptosis to pre-B cells. Overall these studies suffer from the lack of apoptotic positive controls. Further, because of its growth factor independence, the immature B-cell line (2E8) may not be an appropriate model of B-cell apoptosis. Some compounds can alter the apoptotic pathway, it is possible that 1-ethynyl pyrene might alter apoptotic response and this is a weakness that should be addressed. Strengths of this aim include measuring multiple parameters in the apoptotic response (i.e., phosphatidyl serine, laddering and microscopic examination of cellular morphology). The third specific aim characterizes CYP expression and DMBA metabolism in primary bone marrow stromal cells derived from genetically AhR and CYP1B1 deficient mice. The availability of these mice in Dr. Jefcoate's laboratory is a major strength of this aim and these studied should reveal if AhR affects basal and inducible CYP1B1 as well as determine if DMBA metabolism occurs in bone marrow stromal cells in the absence of CYP1B1. The final specific Aim is a logical extension of the previous three aims and will determine if either CYP1B1 or AhR or both are required for B-cell apoptosis by DMBA. Overall, this is a highly focused application and the applicant will clearly benefit from this training. The mentors, Dr. Czuprynski, an expert in immunology and Dr. Jefcoate, an expert in P450 drug metabolism, provide an excellent collaborative interdisciplinary environment to train the candidate.
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|Heidel, S M; MacWilliams, P S; Baird, W M et al. (2000) Cytochrome P4501B1 mediates induction of bone marrow cytotoxicity and preleukemia cells in mice treated with 7,12-dimethylbenz[a]anthracene. Cancer Res 60:3454-60|
|Heidel, S M; Czuprynski, C J; Jefcoate, C R (1998) Bone marrow stromal cells constitutively express high levels of cytochrome P4501B1 that metabolize 7,12-dimethylbenz[a]anthracene. Mol Pharmacol 54:1000-6|