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Gene profiling of cottontail rabbit papillomavirus-induced carcinomas identifies upregulated genes directly Involved in stroma invasion as shown by small interfering RNA-mediated gene silencing.

ABSTRACT: To investigate changes in cellular gene expression associated with malignant progression, we identified differentially expressed genes in a cottontail rabbit papillomavirus (CRPV) squamous carcinoma model employing New Zealand White rabbits. The technique of suppression subtractive cDNA hybridization was applied to pairs of mRNA isolates from CRPV-induced benign papillomas and carcinomas, with each pair derived from the same individual rabbit. The differential expression of 23 subtracted cDNAs was further confirmed by quantitative reverse transcription-PCR (RT-PCR) with additional biopsies. Eight papilloma-carcinoma pairs examined showed a constant upregulation of the transcripts for the multifunctional adaptor protein 14-3-3 zeta and the Y-box binding transcription factor YB-1, whereas transcripts for m-type calpain 2 and NB thymosin beta, which are involved in cell motility and tissue invasion, as well as casein kinase 1 alpha, chaperonin, and annexin I, were found to be upregulated in the majority of the cases. RNA-RNA in situ hybridization and laser capture microdissection in combination with quantitative RT-PCR analysis verified the deregulated expression of the transcripts in the tumor cells. In contrast, CRPV E7 transcript levels remained rather constant indicating no requirement for a further upregulation of E7 expression following tumor induction. Small interfering RNA-mediated interference with expression of genes encoding YB-1, m-type calpain 2, or NB thymosin beta in a CRPV-positive cell line established from New Zealand White rabbit keratinocytes resulted in decreased cell invasion in matrigel chamber assays.

SUBMITTER: Huber E

PROVIDER: S-EPMC434115 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Gene profiling of cottontail rabbit papillomavirus-induced carcinomas identifies upregulated genes directly Involved in stroma invasion as shown by small interfering RNA-mediated gene silencing.

Huber Evamaria E Vlasny Daniela D Jeckel Sonja S Stubenrauch Frank F Iftner Thomas T

Journal of virology 20040701 14

To investigate changes in cellular gene expression associated with malignant progression, we identified differentially expressed genes in a cottontail rabbit papillomavirus (CRPV) squamous carcinoma model employing New Zealand White rabbits. The technique of suppression subtractive cDNA hybridization was applied to pairs of mRNA isolates from CRPV-induced benign papillomas and carcinomas, with each pair derived from the same individual rabbit. The differential expression of 23 subtracted cDNAs w ...[more]

PMID: 15220421

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Project description:BACKGROUND: Recent studies in human have highlighted the importance of the monocyte chemotactic proteins (MCP) in leukocyte trafficking and their effects in inflammatory processes, tumor progression, and HIV-1 infection. In European rabbit (Oryctolagus cuniculus) one of the prime MCP targets, the chemokine receptor CCR5 underwent a unique structural alteration. Until now, no homologue of MCP-2/CCL8a, MCP-3/CCL7 or MCP-4/CCL13 genes have been reported for this species. This is interesting, because at least the first two genes are expressed in most, if not all, mammals studied, and appear to be implicated in a variety of important chemokine ligand-receptor interactions. By assessing the Rabbit Whole Genome Sequence (WGS) data we have searched for orthologs of the mammalian genes of the MCP-Eotaxin cluster. RESULTS: We have localized the orthologs of these chemokine genes in the genome of European rabbit and compared them to those of leporid genera which do (i.e. Oryctolagus and Bunolagus) or do not share the CCR5 alteration with European rabbit (i.e. Lepus and Sylvilagus). Of the Rabbit orthologs of the CCL8, CCL7, and CCL13 genes only the last two were potentially functional, although showing some structural anomalies at the protein level. The ortholog of MCP-2/CCL8 appeared to be pseudogenized by deleterious nucleotide substitutions affecting exon1 and exon2. By analyzing both genomic and cDNA products, these studies were extended to wild specimens of four genera of the Leporidae family: Oryctolagus, Bunolagus, Lepus, and Sylvilagus. It appeared that the anomalies of the MCP-3/CCL7 and MCP-4/CCL13 proteins are shared among the different species of leporids. In contrast, whereas MCP-2/CCL8 was pseudogenized in every studied specimen of the Oryctolagus - Bunolagus lineage, this gene was intact in species of the Lepus - Sylvilagus lineage, and was, at least in Lepus, correctly transcribed. CONCLUSION: The biological function of a gene was often revealed in situations of dysfunction or gene loss. Infections with Myxoma virus (MYXV) tend to be fatal in European rabbit (genus Oryctolagus), while being harmless in Hares (genus Lepus) and benign in Cottontail rabbit (genus Sylvilagus), the natural hosts of the virus. This communication should stimulate research on a possible role of MCP-2/CCL8 in poxvirus related pathogenicity.

Project description:Sylvilagus floridanus Papillomavirus (SfPV) causes growth of large horn-like tumors on rabbits. SfPV was described in cottontail rabbits (probably Sylvilagus floridanus) from Kansas and Iowa by Richard Shope in 1933, and detected in S. audubonii in 2011. It is known almost exclusively from the US Midwest. We explored the University of Kansas Natural History Museum for historical museum specimens infected with SfPV, using molecular techniques, to assess if additional wild species host SfPV, and whether SfPV occurs throughout the host range, or just in the Midwest. Secondary aims were to detect distinct strains, and evidence for strain spatio-temporal specificity. We found 20 of 1395 rabbits in the KU collection SfPV symptomatic. Three of 17 lagomorph species (S. nuttallii, and the two known hosts) were symptomatic, while Brachylagus, Lepus and eight additional Sylvilagus species were not. 13 symptomatic individuals were positive by molecular testing, including the first S. nuttallii detection. Prevalence of symptomatic individuals was significantly higher in Sylvilagus (1.8%) than Lepus. Half of these specimens came from Kansas, though new molecular detections were obtained from Jalisco-Mexico's first-and Nebraska, Nevada, New Mexico, and Texas, USA. We document the oldest lab-confirmed case (Kansas, 1915), pre-dating Shope's first case. SfPV amplification was possible from 63.2% of symptomatic museum specimens. Using multiple methodologies, rolling circle amplification and, multiple isothermal displacement amplification in addition to PCR, greatly improved detection rates. Short sequences were obtained from six individuals for two genes. L1 gene sequences were identical to all previously detected sequences; E7 gene sequences, were more variable, yielding five distinct SfPV1 strains that differing by less than 2% from strains circulating in the Midwest and Mexico, between 1915 and 2005. Our results do not clarify whether strains are host species specific, though they are consistent with SfPV specificity to genus Sylvilagus.

Dataset Information

Gene profiling of cottontail rabbit papillomavirus-induced carcinomas identifies upregulated genes directly Involved in stroma invasion as shown by small interfering RNA-mediated gene silencing.

Publications

Gene profiling of cottontail rabbit papillomavirus-induced carcinomas identifies upregulated genes directly Involved in stroma invasion as shown by small interfering RNA-mediated gene silencing.

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