Alterations in gene expression associated with the overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, during human hepatocarcinogenesis

Abstract

Purpose: Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, correlates significantly with DNA hypomethylation in pericentromeric satellite regions, which is known to result in centromeric decondensation and enhanced chromosomal recombination in precancerous conditions and hepatocellular carcinomas (HCCs). We aimed to elucidate further the significance of DNMT3b4 during human hepatocarcinogenesis.

Methods: DNMT3b4-transfected human epithelial 293 cells were characterized using growth rate measurements, gene expression microarray, and quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses. RT-PCR was also performed on eight normal liver specimens, 45 noncancerous liver specimens showing chronic hepatitis or cirrhosis, which are considered to be precancerous conditions, and 56 HCCs.

Results: The growth rate of the DNMT3b4 transfectants was about double that of mock-transfectants. Induction of signal transducer and activator of transcription 1 (STAT1), an effector of interferon signaling, and of a set of downstream genes implicated in such signaling, was observed in the DNMT3b4 transfectants. There was significant correlation between the mRNA expression levels of DNMT3b4 and STAT1 in HCCs. mRNA expression levels of STAT1 and the three downstream genes examined were all significantly elevated in the chronic hepatitis and cirrhosis specimens compared with the normal liver specimens. Among the HCCs, the mRNA expression levels of STAT1 and the downstream genes were higher in tumors without portal vein involvement than in more malignant HCCs with portal vein involvement. Significant correlations between the mRNA expression levels of STAT1 and each of the downstream genes were observed in the tissue samples.

Conclusions: Overexpression of DNMT3b4 is involved in human hepatocarcinogenesis, even at the precancerous stages, not only by inducing chromosomal instability but also by affecting the expression of specific genes.

Fig. 1

Growth curves for the DNMT3b4 transfectants (clones 3b4–4 and 3b4–5) and mock-transfectants (clones mock-1 and mock-2). Full-length human DNMT3b4 cDNA was transfected into 293 cells during our previous study (Saito et al. 2002). About 50 days after transfection, 3×10⁵ cells were plated into 6-well microtiter plates. Two wells were trypsin-treated at each time point (3 days, 6 days, 8 days, 10 days, and 12 days) and the cells were counted. Open circles mock-1, open squares mock-2, solid circles 3b4–4, solid squares 3b4–5

Fig. 2

Results of the quantitative RT-PCR analysis of STAT-1 in DNMT3b4 transfectants (clones 3b4–4 and 3b4–5) and mock-transfectants (clones mock-1 and mock-2). Full-length human DNMT3b4 cDNA was transfected into 293 cells during our previous study (Saito et al. 2002). All examinations were performed using the GeneAmp 5700 Sequence Detection System (Applied Biosystems) as described in the Materials and Methods section. cDNAs derived from the HCC cell line Alexander were used as calibration samples. The mRNA levels for each clone are expressed as a ratio relative to GAPDH mRNA

Fig. 3A–E

Results of quantitative RT-PCR analyses of A DNMT3b4, B STAT1, C interferon-stimulated gene 12 (GenBank accession number: X67325), D interferon-inducible protein 9-27 (J04164), and E interferon-inducible 56 kD protein (M24594) in normal liver tissue specimens (N), noncancerous liver tissue specimens showing chronic hepatitis (CH), noncancerous liver tissue specimens showing cirrhosis (LC) and HCCs. (W well-differentiated HCCs, M moderately differentiated HCCs, P poorly differentiated HCCs, − portal vein involvement-negative, + portal vein involvement-positive). For eight normal liver specimens, 45 noncancerous liver specimens showing chronic hepatitis or cirrhosis, and 50 HCCs included in this study, expression levels of DNMT3b4 mRNA had already been evaluated by quantitative RT–PCR and reported in our previous study (Saito et al. 2002). In panel A, correlation between the previously reported DNMT3b4 mRNA expression levels, and clinicopathological parameters of tissue samples was reexamined for comparison with panels B–E. As we have already reported (Saito et al. 2002), DNMT3b4 expression was especially elevated in precancerous liver tissues with marked DNA hypomethylation of pericentromeric satellite regions. Therefore, the difference in the average levels of DNMT3b4 expression between all CH and LC with and without DNA hypomethylation in these regions and N did not reach a statistically significant level (A), although DNMT3b4 showed a generally similar pattern of expression alteration to that of STAT1 and other genes implicated in interferon signaling shown in panels B–E. Other examinations were performed using the GeneAmp 5700 Sequence Detection System (Applied Biosystems) as described in the Materials and Methods section. cDNAs derived from the HCC cell line Alexander or the stomach cancer cell line MKN1 were used as calibration samples. The mRNA levels for each sample are expressed as the ratio relative to GAPDH mRNA

Fig. 4A,B

A Correlation between the mRNA expression levels of DNMT3b4 and STAT1 in HCCs and B correlations between the mRNA expression levels of STAT1 and each of the downstream genes [interferon-stimulated gene 12 (GenBank accession number: X67325), interferon-inducible protein 9–27 (J04164) and interferon-inducible 56 kD protein (M24594)] in normal liver specimens, chronic hepatitis specimens, cirrhosis specimens, and HCCs. For 50 HCCs presented in panel A, DNMT3b4 mRNA expression levels had already been evaluated by quantitative RT–PCR and reported in our previous study (Saito et al. 2002). Other examinations were performed using the GeneAmp 5700 Sequence Detection System (Applied Biosystems) as described in the Materials and Methods section. cDNAs derived from the HCC cell line Alexander or the stomach cancer cell line MKN1 were used as calibration samples. The mRNA levels for each sample are expressed as the ratio relative to GAPDH mRNA