Retinoids and Retinoid Metabolism in Breast Cancer.
Annual rept. 16 Jun 97-15 Jun 98
WASHINGTON UNIV SEATTLE
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My studies aim to understand vitamin A metabolism in breast tissue, specifically the role of the human 9 cis-retinol dehydrogenase 9cRDH. The 9cRDH gene was cloned from a normalized mammary library by Blaner and colleagues. We studied its expression in two normal human mammary epithelial cell strains HMECs and a panel of breast cancer cell lines. mRNA levels were determined by a semi-quantitative, multiplex reverse transcriptase-polymerase chain reaction RT-PCR strategy. 9cRDH mRNA levels were similar among the cells tested, with the exception of the breast cancer cell lines, Hs578T and T47D, which exhibited higher expression than the normal strains, and MCF-7, which showed the lowest levels. Individual 9cRDH activity levels in normal and tumor cells, as measured by normal phase HPLC, paralleled expression levels observed by RT-PCR. In order to study the biochemical characteristics of 9cRDH and to determine optimal activity test conditions, we developed a Hep G2 cell line that expresses high levels of 9cRDH protein LRDHSNHep G2. The 9cRDH cDNA was transduced into the cells using the LXSN retroviral vector containing the full length 9cRDH gene. The activity of 9cRDH tested in microsomes of LRDHSNHep G2 cells was time- and protein concentration-dependent and followed Michaelis-Menten kinetics.
- Medicine and Medical Research