Human hepatocellular carcinoma expresses specific PCNA isoforms: an in vivo and in vitro evaluation

Abstract

Proliferating cell nuclear antigen (PCNA) is a 36 kDa protein involved in several cellular mechanisms, including DNA synthesis and repair, cell cycle regulation and apoptosis. An alteration in PCNA structure might contribute to DNA-damage accumulation in cancer cells. This study was aimed to evaluate the PCNA pattern of expression, in terms of aggregation status, isoforms and post-translational modifications, in human hepatocellular carcinoma (HCC) and cirrhosis as well as in HCC cell lines. Twelve HCCs and surrounding cirrhotic tissues were analysed, along with HepG2, Hep3B and SNU-398 cell lines. Normal liver specimens and cirrhosis without HCC were included as controls. Both DNA-bound and DNA-unbound PCNA fractions were analysed, and PCNA pattern of expression was displayed on two-dimensional gel electrophoresis followed by western blot. Results were confirmed by mass spectrometry. To compare HCCs vs surrounding tissues, immunolabelling and immunostaining were performed. In 6 of 12 HCCs and in cell lines, we found three major PCNA acidic forms, corresponding to monomers, probably dimers and trimers, and a basic isoform. In the six remaining HCCs, only a PCNA acidic form associated with multiple basic isoforms was detected. Importantly, the PCNA basic form was not found in cirrhotic tissues. To clarify the nature of the detected PCNA isoforms, ubiquitin-specific immunoblotting as well as phosphatase treatment were employed. A PCNA-ubiquitylated form in cell lines and PCNA-phosphorylated isoforms in 6 of 12 HCCs were detected. Finally, in the DNA-bound fraction we detected only an acidic PCNA monomeric form. We conclude that human hepatocellular carcinoma expresses specific PCNA isoforms compared to those found in cirrhosis, implicating a role for PCNA functional alterations in hepatocarcinogenesis.

A Peptide Mimicking a Region in Proliferating Cell Nuclear Antigen Specific to Key Protein Interactions Is Cytotoxic to Breast Cancer

“…These malignant cells harbor an additional isoform of PCNA with an acidic pI, as opposed to the normal cells, which only contain PCNA with a basic pI. Similar PCNA profiles are present in other types of cancer, including neuroblastoma (Sandoval et al, 2005), hepatic carcinoma (Venturi et al, 2008), and high-grade prostatic intraepithelial neoplasia and prostate cancer (Wang et al, 2011). The newly identified cancer-associated acidic isoform of PCNA (caPCNA) results from a set of post-translational modifications .…”

Validating the disruption of proliferating cell nuclear antigen interactions in the development of targeted cancer therapeutics

“…9 Two-dimensional PAGE analysis illustrated that malignant breast cells harbored an additional isoform of PCNA with an acidic isoelectric point (pI), as opposed to the normal cell expressing PCNA with only a basic pI. The two distinct PCNA isoforms have been resolved in a variety of cancer cell lines, including neuroblastoma, 10 hepatic carcinoma, 11 high-grade prostatic intraepithelial neoplasia and prostate cancer. 12 The acidic isoform associated with cancer (caPCNA) is not the result of genetic mutations or alternate splicing, but is the result of differential post-translational modification within the PCNA molecule.…”

Potential Antitumor Activity and Apoptosis Induction of Glossostemon bruguieri Root Extract against Hepatocellular Carcinoma Cells

“…Venturi et al reported the detection of two PCNA expression patterns in human liver tumors and cell lines [70]. The different pools of PCNA might act together and at the same time to carry out different functions within different signaling pathways, as reported previously [71].…”

“…Taken together, during cell cycle arrest, PCNA protein may form complexes with several proteins (p21, p57, Gadd45, MyD118, CR6, ING1, MCL1, MutS α , and MutL α ). PCNA functions and interactions are modulated by posttranslational regulation which is thought to guide PCNA to the correct partner protein at the correct time to perform a specific function [70]. Studies suggest that PCNA is the supervisory molecule in life of the cell, forcing it to replicate DNA, arrest the cell in G1 or G2 phase, repair damaged DNA, or obligate the cell to apoptosis, based on the interactions with a plethora of proteins [72].…”