Attempts at immortalization of crustacean primary cell cultures using human cancer genes

Abstract

Primary cell cultures from crustacea have been initiated since the 1960s, yet no permanent cell line is available. Primary cells have a limited proliferative capacity in culture due to cellular senescence, which is regulated by a group of dominant senescence genes. The aim of this research was to manipulate cell cycle regulation by transfecting Cherax quadricarinatus primary cells with oncogenes, in an effort to induce a permanent cell line. Human papillomaviruses (HPV) play a critical role in the formation of anogenital cancer. Research has demonstrated that the HPV-expressed E6 and E7 proteins function concomitantly to disrupt the p53 and retinoblastoma (Rb) tumor suppressor genes, regulators of the cell-cycle checkpoints at the first gap (G(1)) phase. HPV E6 and E7 genes were transfected into the C. quadricarinatus cells by lipofection. Successful transfection was demonstrated by the presence of oncogene messenger RNA by reverse transciptase polymerase chain reaction. At day 150, transfected cells still remain viable, although cell proliferation was stagnant. It may be that while transfection of the oncogenes was successful, no proliferation of the C. quadricarinatus cells was evident due to a lack of telomere maintenance.

Establishment of Shrimp Cell Lines: Perception and Orientation

“…Given the tremendous advancements in human and veterinary virology thanks to the availability of a variety of cell lines, any radical change in crustacean virology would be possible only if appropriate cell lines for in vitro cultivation of intracellular pathogenic agents [17] could be made available. Considering the past experience in this realm more focus should be on the molecular approaches to immortalize shrimp cells by disrupting cell cycle regulator genes and the telomere maintenance.…”

“…The high regeneration capability of the crustacean cells (including shrimp) do not show neoplastic transformation and thus it prevents spontaneous immortalization. Neoplastic transformation can be achieved by transfection with active oncogenes [66], the technique which has not yet been fully applied to crustacean and aquatic invertebrate cells [17]. Moreover, unveiling the molecular and regulatory mechanisms that prevent neoplastic transformation in shrimp cells (decapod crustaceans) might provide new leads for the development of anti-ageing and anti-cancer interventions in humans [86].…”

“…Even though, researchers [36,37] proved the use of VSV-G pseudotyped pantropic retroviral vectors by confirming the stable expression of SV40T gene in post transfected cells, the attempts failed to induce in vitro transformation. Moreover, Claydon and Owens [17] transfected human papillomaviruses (HPV) E6 and E7 genes into the Cherax quadricarinatus cells by lipofection and the successful transfection was demonstrated by the presence of oncogene mRNA by RT-PCR. While transfection of the oncogenes was successful and transfected cells survived more than 150 days, cell proliferation was stagnant due to the lack of telomere maintenance.…”

Susceptibility of testicular cell cultures of crab, Scylla serrata (Forskal) to white spot syndrome virus

“…Although new information is available on WSSV, relatively less is known about host parasite interactions (Jiravanichpaisal et al 2006). To understand and ultimately to control the viral diseases in crustacea, a specific and sensitive diagnostic tool to investigate this pathogen needs to be developed (Claydon and Owens 2008).…”

“…Claydon and Owens (2008) reported that due to the presence of dominant senescence genes, crustacean primary cell culture has limited proliferations. A few primary cultures of crayfish and prawn tissues have been developed earlier for the diagnosis of viruses infecting crustaceans (Uma et al 2002;Jiravanichpaisal et al 2006;Li and Shields 2007;Seena et al 2010) but long surviving and proliferating cell cultures were not available for evaluating viral infections.…”