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Expression of Tumor-associated Aldehyde Dehydrogenase Gene in Rat Hepatoma Cell Lines¹

Department of Biology, The University of Alabama, Tuscaloosa, Alabama 35487

Significant changes in aldehyde dehydrogenase (ALDH) activity occur during rat hepatocarcinogenesis in vivo. To compare the structure and expression of the tumor aldehyde dehydrogenase gene in rat hepatoma cell lines and normal rat liver, several rat hepatoma cell lines, including HTC, H4-II-EC₃, JM₂, McA-RH7777, and four lines established in this laboratory have been examined for T-ALDH gene expression using a tumor ALDH complementary DNA.

Northern blot analysis of polyadenylate-containing RNA from logphase cells and normal rat liver with T-ALDH complementary DNA indicates production of a single major 1.7-kilobase transcript in the high activity lines HTC, JM₂, RLT-2M, RLT-3C, RLT-9F, and intermediate activity line RLT-5G. There is a direct correlation between expression of T-ALDH enzyme activity and the amount of 1.7-kilobase transcript. S₁ nuclease protection experiments confirm that there is only one major T-ALDH transcript in the high activity lines. Thus, cell line differences in T-ALDH activity are reflected in the level of a single T-ALDH transcript.

Southern analysis was used to identify the T-ALDH gene in genomic DNA. The results indicate that no significant amplification or rearrangement of the T-ALDH gene has occurred in these hepatoma cells. DNA methylation has been proposed to play an important role in gene expression. Genomic DNA from HTC, JM₂, McA-RH7777, H4-II-EC₃, RLT-2M, RLT-9F, RLT-3C, RLT-5G, rat embryo and normal rat liver were digested with MspI and HpaII to examine methylation patterns. A digestion pattern consistent with hypomethylation was detected only in DNA from the high T-ALDH activity cell lines HTC, JM₂, RLT-2M, and RLT-9F. This suggests that constitutive expression of T-ALDH in the hepatoma cells is related to changes in DNA methylation patterns.

¹ Supported by University of Alabama Graduate Council Research Fellowship to K-h. L., NIH Grant CA-21103 to R. L., and University of Alabama funds to M. D. B.

² Submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in The Graduate School of The University of Alabama. Present address: Building 37, Room 3C20, NIH, Bethesda, MD 20892.

³ To whom requests for reprints should be addressed.

Received 10/ 9/87. Revised 7/25/88. Accepted 9/14/88.