Cytochrome P450 1B1 mRNA Research Articles

Regulation of cytochrome P4501B1 in cultured rat adrenocortical cells by cyclic adenosine 3',5'-monophosphate and 2,3,7,8- tetrachlorodibenzo-p-dioxin.

Cytochrome P4501B1 (CYP1B1), which is responsible for metabolism of 7,12-dimethylbenz[a]anthracene in the rat adrenal gland, is partially dependent on ACTH in vivo. The regulation of CYP1B1 and possible involvement in steroidogenesis have been characterized in cultured rat adrenocortical cells. Relatively high basal expression of CYP1B1 is maintained in vitro and is, therefore, independent of ACTH. CYP1B1 expression is elevated 4-fold in primary cultures of fasciculata cells after 24 h of ACTH treatment, as measured by selective 7,12-dimethylbenz[a]anthracene metabolism, immunoblot analysis, and parallel changes in the 5.2-kilobase CYP1B1 messenger RNA (mRNA). Corticosterone synthesis was stimulated about 40-fold in these cells after this ACTH treatment. Maximal stimulation of CYP1B1 protein and mRNA by ACTH has been duplicated in fasciculata cells by 8-bromo-cAMP and the adenylyl cyclase agonist, forskolin, indicating that cAMP mediates this induction. CYP1B1 is similarly stimulated by ACTH in rat adrenal glomerulosa cells, although constitutive expression of CYP1B1 is about 4-fold lower. Angiotensin II treatment of glomerulosa cells, which stimulated aldosterone synthesis 3-fold, had no effect on CYP1B1 activity or expression. Treatment of fasciculata and glomerulosa cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in small increases in CYP1B1 activity (1.8- and 2.5-fold, respectively), but much larger increases (5- and 6-fold, respectively) in CYP1B1 at the mRNA level 2,3,7,8-Tetrachlorodibenzo-p-dioxin had no effect in the presence of ACTH stimulation. CYP1B1 is not coordinately expressed with steroidogenic enzymes. CYP11A1 and CYP21 mRNAs are far more responsive to ACTH, in part because of lower basal expression. CYP1B1 exhibited a transient response to ACTH that peaked (9-fold) at 6 h before declining to about 4-fold at 36 h, the time when CYP21 mRNA was maximally stimulated. The complete inactivation of CYP1B1 activity in fasciculata cells by a mechanism-based inhibitor, 1-ethynylpyrene, did not affect corticosterone production, indicating that this protein does not have a direct physiological role in the steroidogenic response.

Lack of correlation between expression of Epstein-Barr virus (EBV) latent membrane protein and bcl-2 oncoprotein in vivo.

To evaluate whether there is any correlation between the expression of Epstein-Barr virus (EBV) latent membrane protein (LMP) and oncoprotein bcl-2 in the lymph node biopsy specimens of a Chinese patient with EBV-related reactive lymphoproliferation who later developed T cell lymphoma after a short period of time. Immunohistochemistry, with a standard alkaline phosphatase antialkaline phosphatase (APAAP) method and New Fuchsin as a chromogen, was used for single staining of bcl-2 or LMP. Double immunostaining combining APAAP and indirect immunofluorescence was performed for dual labelling of LMP and bcl-2. bcl-2 was expressed in 10-30% of cells in the first lymph node biopsy specimen (EBV-associated lymphoproliferative disorder) and 30-50% of cells in the second lymph node biopsy specimen (T cell lymphoma). LMP was expressed in the first biopsy specimen but not in the second. Double immunostaining results showed that around 78% of LMP positive cells were bcl-2 negative and 94% bcl-2 positive cells were LMP negative. Among the very small fraction of LMP and bcl-2 double positive cells, the intensity of bcl-2 staining was heterogeneous and was not always stronger than that observed in LMP negative bcl-2 positive cells. The expression of bcl-2 protein is independent of LMP protein status in vivo. Several mechanisms may be involved in EBV associated lymphomagenesis, and bcl-2 induction may occur independently of LMP expression.

Complete cDNA sequence of a human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2.

Previously, levels of a novel human mRNA, detected by a recombinant cDNA designated clone 1, were shown to be increased 50-fold in response to treatment of a keratinocyte cell line with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in part as a function of increased rates of gene transcription (Sutter, T.R., Guzman, K., Dold, K.M., and Greenlee, W.F. (1991) Science 254, 415-418). Here we report the complete corresponding 5.1-kilobase cDNA sequence. A single open reading frame that predicts a protein of 543 amino acid residues was determined by computer-assisted analysis of the cDNA sequence. This predicted protein identifies a new gene subfamily of cytochrome P450, cytochrome P4501B1 (CYP1B1), that maps to human chromosome 2. Southern blot analysis of genomic DNA indicates that the human CYP1B subfamily is likely to contain only this single gene. Northern blot analysis of RNA isolated from primary cultures of normal human epidermal keratinocytes showed approximately 100-fold increased levels of the CYP1B1 mRNA after treatment with 10 nM TCDD for 24 h. Low levels of constitutive CYP1B1 mRNA were detected in 15 different human tissue samples. These results indicate that CYP1B1 is expressed in many normal human tissues and advance our understanding of the complexity of a gene family of cytochromes P450 whose expression is altered by TCDD.