Elsevier

Human Pathology

Volume 36, Issue 11, November 2005, Pages 1154-1162
Human Pathology

Original contribution
GADD45-α expression in cirrhosis and hepatocellular carcinoma: relationship with DNA repair and proliferation

https://doi.org/10.1016/j.humpath.2005.07.017Get rights and content

Summary

Growth arrest and DNA damage 45-α (GADD45-α) is a nuclear protein involved in maintenance of genomic stability, DNA repair, and suppression of cell growth through interaction with nuclear elements, including cyclin-dependent kinase inhibitor 1A (CDKN1A) and PCNA. In this study, GADD45-α expression was assessed in 28 cases of hepatocellular carcinoma (HCC) and matched cirrhosis tissues, and correlated with the presence of DNA-bound PCNA and CDKN1A as markers of DNA repair, as well as with clinicopathologic variables including histopathologic grade, tumor size, nodularity, viral status, α-fetoprotein serum levels, and p53 and Ki67 immunostaining. GADD45-α and CDKN1A messenger RNA (mRNA) were analyzed by reverse transcriptase–polymerase chain reaction. GADD45-α protein expression was evaluated by Western blot (WB) and enzyme-linked immunosorbent assays (ELISAs). PCNA and CDKN1A DNA-bound fractions were determined by WB. GADD45-α mRNA was down-regulated in 20 of 26 HCCs with respect to matched cirrhosis, but no correlation was found with the corresponding protein levels assessed by both WB and ELISA. GADD45-α and CDKN1A protein levels were related to each other both in cirrhotic and in neoplastic tissues, and a concordant up- or down-regulation was observed in HCCs with respect to cirrhosis. DNA-bound PCNA and CDKN1A were present in 5 HCCs and were associated with higher GADD45-α protein levels assessed by ELISA. No significant association was found in HCCs between GADD45-α protein expression and histopathologic grading, nodule size, focality, and proliferation, whereas a positive correlation was found with α-fetoprotein serum levels.

In conclusion, GADD45-α mRNA was down-regulated with respect to matched cirrhosis in most HCCs; however, no correlation was found between mRNA and protein levels. GADD45-α protein levels were higher in HCCs with DNA-bound CDKN1A and PCNA, suggesting a possible role in DNA repair.

Introduction

Growth arrest and DNA damage 45-α (GADD45-α) is an 18-kd nuclear protein induced upon DNA damage in a p53-dependent and p53-independent fashion [1], [2]. It is a cell cycle–regulated protein that reaches its maximal expression levels in the G1 phase, and like the checkpoint protein cyclin-dependent kinase inhibitor 1A (CDKN1A/p21/cip1/sdi1/waf1, hereafter referred to as CDKN1A), it decreases during the G1/S phase transition [3]. In the experimental models, GADD45-α expression inhibits the entry in the S phase, perhaps by displacing cyclin D1 from its interaction with PCNA from the quaternary complex [4]. Moreover, it induces cell cycle arrest [5] and reduces cell growth primarily through activation of the G2 checkpoint in the cell cycle [6]. Recent studies have suggested a direct role of GADD45-α in the control of apoptosis [7], maintenance of genomic stability [8], and regulation of signaling pathways [9].

GADD45-α co-immunoprecipitates with PCNA [4], [10] with growing experimental evidence, supporting its role in the repair of damaged DNA [4], [11], [12], [13], [14], [15]. GADD45-α was also shown to interact with CDKN1A [3], [16], and it was suggested that it could sequester CDKN1A during the initial steps of repair, thus allowing the repair process itself [17]. Recent findings suggest that a significant part of cellular GADD45-α is strictly bound to the insoluble nuclear matrix/chromatin fraction, particularly in response to DNA damage [18]. In DNA-repairing conditions, therefore, GADD45-α is only partially extractable from the cells, and analysis by Western blot (WB) could be misleading.

Genomic instability and DNA damage have been extensively demonstrated both in cirrhotic and in hepatocellular carcinoma (HCC) tissues [19], [20], [21]. During DNA repair, a fraction of PCNA and CDKN1A relocate to repair sites, tightly bind DNA, and become detergent insoluble [22], [23]. We have recently shown in HCC tissue the presence of DNA-bound fractions of CDKN1A and PCNA, highly suggestive for ongoing DNA-repair activity [24].

To our knowledge, no data are available on GADD45-α expression in HCC and cirrhosis. Because in vitro studies have shown that GADD45-α is up-regulated after DNA damage, our work aims to verify if GADD45-α modulation occurs also in vivo in pathological conditions with well-documented DNA damage. To test this hypothesis, we have analyzed GADD45-α both at messenger RNA (mRNA) and protein levels in a series of resected HCCs and cirrhosis, including cases with and without ongoing DNA repair activity as determined by the presence of DNA-bound fractions of PCNA and CDKN1A. GADD45-α expression was also correlated with tumor characteristics (histopathologic grade, size, nodularity, viral status, and α-fetoprotein [AFP] serum levels), p53 immunostaining, and cell growth fraction, and evaluated by Ki67 labeling index (LI).

Section snippets

Patients

Twenty-eight patients undergoing partial hepatectomy for HCC prospectively entered the study. The inclusion criteria were the preservation of frozen tissue from at least one of the HCC nodules obtained from surgery and the exclusion of previous local or systemic anticancer treatment. Informed consent was obtained from each patient before surgery, which consisted of partial hepatectomy in 27 cases and orthotopic liver transplantation in 1 case (Table 1, patient 3). The study protocol conforms to

GADD45-α and CDKN1A mRNA and protein expression

GADD45-α mRNA turned out to be down-regulated in 20 of 26 HCCs (76.9%) with respect to matched cirrhosis, whereas the remaining 6 cases displayed an up-regulation. For both CDKN1A and GADD45-α, no significant correlation was found between mRNA and protein expression. When up- or down-regulation of GADD45-α mRNA and the corresponding protein (assessed by ELISA and WB) were analyzed in HCC and matched cirrhosis, again, no association was found (Fisher exact test, P = .400 and P = .124,

Discussion

This study provides the first evidence of GADD45-α mRNA and protein expression in cirrhotic and HCC tissues. Our results have shown an association between GADD45-α protein expression and the presence of a DNA-bound fraction of PCNA and CDKN1A, in line with its possible involvement in DNA repair. No previous data are reported in the literature concerning GADD45-α expression in liver diseases, where, however, genomic instability and DNA damage have been extensively reported [19], [20], [21].

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    This work was supported by funds from Fondazione Cassa di Risparmio in Bologna and Ministry of Instruction, University and Research (MIUR).

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