Isolation of the Retinoblastoma cDNA from the Marine Flatfish Dab (Limanda limanda) and Evidence of Mutational Alterations in Liver Tumors

Abstract

We have isolated a dab (Limanda limanda) homologue of the human retinoblastoma (Rb) tumor suppressor gene. The L. limanda partial Rb cDNA encodes a partial predicted protein of 753 amino acids. DNA sequence analysis with other vertebrate Rb sequences demonstrates that the L. limanda Rb cDNA is highly conserved in regions of functional importance. The sequence reported herein, combined with the high degree of conservation observed in critical domains, has also facilitated an investigation of the molecular etiology of environmentally induced liver tumor samples in a feral fish species. Mutational alterations were detected in liver adenoma samples, also in apparently "normal" regions of liver samples dissected from fish displaying adenoma, but not in normal liver samples from otherwise healthy feral fish. These results are the first reporting the appearance of Rb mutations in wild-caught fish and suggest that the molecular etiology of fish cancer appears to involve Rb-implicated tumorigenesis. The ecotoxicological relevance of the Rb mutations in feral fish liver tumors, in terms of future genome instability and possible development of a genotoxicity biomarker, is discussed.

Gene expression analyses of hepatocellular adenoma and hepatocellular carcinoma from the marine flatfish Limanda limanda

“…The expression changes that are likely to occur in and between tumor sub-types may well provide the foundation for molecular classification of tumors, and provide prognostic markers that can be used as indicators of pollution-induced damage. In addition, utilization of tumor tissues from wild animals will provide additional useful information for comparison with laboratory-induced tumors, since this will likely reflect the multi-factorial nature of cancer formation in natural populations (du Corbier et al 2005, Ward et al 2006. DNA microarrays have been applied in many areas of biological research, including human cancer (Rhodes et al 2004), and are increasingly being used in studies of gene expression in fish (Douglas 2006), primarily as a tool for understanding development processes and basic physiology.…”

Hepatocellular adenoma in a European flatfish (Limanda limanda): Genetic alterations in laser-capture micro-dissected tissue and global transcriptomic approach

“…Such lesions have been associated with exposure to anthropogenic contaminants such as polycyclic aromatic hydrocarbons (PAHs) (Malins et al, 1985;Vethaak and Reinallt, 1992). Dab possess both a similar histopathological tumour profile to humans (Stern and Zon, 2003) and homologs of human cancer genes such as ras and retinoblastoma (Rb), including mutational alterations of the Rb gene in tumour tissues (Du Corbier et al, 2005). In this respect, we have previously proposed that the dab hepatocellular tumour development could act as surrogate for cancer and the tumourigenesis process in human populations (Rotchell et al, 2009).…”

“…This diagnostic approach has recently been coupled with molecular analyses of tumour and surrounding normal tissue (Small et al, 2010). In addition, gross lesions and apparently normal tissues have been resected from the dab liver for molecular investigations such as genetic alterations of neoplastic associated genes (Du Corbier et al, 2005;Rotchell et al, 2009, Lerebours et al, 2014, transcriptomic (Small et al, 2010), proteomic (Stentiford et al, 2005) and metabolomic studies (Stentiford et al, 2005;Southam et al, 2008). Traditional microdissection and molecular analysis of populations of cells presents a challenge in that cellular heterogeneity within tissues samples may result in misleading findings (Cole et al, 1999;Sluka et al, 2008).…”

“…Rb was selected on the basis that Rb mutations have previously been reported in dab liver tumour samples (Du Corbier et al, 2005;Lerebours et al, 2014). In parallel, a global transcriptomic approach using the suppressive subtractive hybridisation (SSH) method was used to analyse the differentially expressed genes between HCA-bearing liver and normal liver samples.…”

Advanced diagnostics applied to fish liver tumours: Relating pathology to underlying molecular aetiology

“…Such lesions have been associated with exposure to anthropogenic contaminants such as polycyclic aromatic hydrocarbons (PAHs) (Malins et al, 1985;Vethaak and Reinallt, 1992). Dab possess both a similar histopathological tumour profile to humans (Stern and Zon, 2003) and homologs of human cancer genes such as ras and retinoblastoma (Rb), including mutational alterations of the Rb gene in tumour tissues (Du Corbier et al, 2005).…”