Inducer Effect on the Complex Formation between Rat Liver Nuclear Proteins and Cytochrome P450 2B Gene Regulatory Elements

T. G. Duzhak, E. I. Schwartz, L. F. Gulyaeva^*, and V. V. Lyakhovich

Institute of Molecular Biology and Biophysics, Siberian Branch of the Russian Academy of Medical Sciences, ul. Timakova 2, Novosibirsk, 630117 Russia; fax: (383-2) 323-147; E-mail: gulyaeva@cyber.ma.nsc.ru; gulyaeva@hotmail.com

^* To whom correspondence should be addressed.

Received September 14, 2001; Revision received February 27, 2002

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DNA gel retardation assay has been applied to the investigation of complexes between rat liver nuclear proteins and Barbie box positive regulatory element of cytochrome P450 2B (CYP2B) genes. The intensities of B1 and B2 bands detected in the absence of an inducer increased after 30 min protein incubation with phenobarbital (PB) or triphenyldioxane (TPD), but not with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOPOB). In addition, a new complex (B3 band) was for the first time detected under induction by PB, TPD, and TCPOPOB. Increase in the incubation time up to 2 h facilitated the formation of other new complexes (B4 and B5 bands), which were detected only in the presence of TPD. The use of [³H]TPD in hybridization experiments revealed that this inducer, capable of binding to Barbie box DNA, is also present in B4 and B5 complexes. It is probable that the investigated compounds activate the same proteins at the initial induction steps, which correlates with the formation of B1, B2, and B3 complexes. The further induction step might be inducer-specific, as indicated by the formation of B4 and B5 complexes in the presence of TPD only. Thus, the present data suggest the possibility of specific gene activation signaling pathways that are dependent on a particular inducer.

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KEY WORDS: rat CYP2B2 gene, triphenyldioxane, phenobarbital, Barbie box, nuclear proteins, gel retardation assay

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