Transcription from a gene desert in a melanoma porcine model.

The genetic mechanisms underlying cutaneous melanoma onset and progression need to be further understood to improve patients' care. Several studies have focused on the genetic determinism of melanoma development in the MeLiM pig, a biomedical model of cutaneous melanoma. The objective of this study was to better describe the influence of a particular genomic region on melanoma progression in the MeliM model. Indeed, a large region of the Sus scrofa chromosome 1 has been identified by linkage and association analyses, but the causal mechanisms have remained elusive. To deepen the analysis of this candidate region, a dedicated SNP panel was used to fine map the locus, downsizing the interval to less than 2 Mb, in a genomic region located within a large gene desert. Transcription from this locus was addressed using a tiling array strategy and further validated by RT-PCR in a large panel of tissues. Overall, the gene desert showed an extensive transcriptional landscape, notably dominated by repeated element transcription in tumor and fetal tissues. The transcription of LINE-1 and PERVs has been confirmed in skin and tumor samples from MeLiM pigs. In conclusion, although this study still does not identify a candidate mutation for melanoma occurrence or progression, it highlights a potential role of repeated element transcriptional activity in the MeLiM model.

Rapid Discovery of De Novo Deleterious Mutations in Cattle Enhances the Value of Livestock as Model Species.

In humans, the clinical and molecular characterization of sporadic syndromes is often hindered by the small number of patients and the difficulty in developing animal models for severe dominant conditions. Here we show that the availability of large data sets of whole-genome sequences, high-density SNP chip genotypes and extensive recording of phenotype offers an unprecedented opportunity to quickly dissect the genetic architecture of severe dominant conditions in livestock. We report on the identification of seven dominant de novo mutations in CHD7, COL1A1, COL2A1, COPA, and MITF and exploit the structure of cattle populations to describe their clinical consequences and map modifier loci. Moreover, we demonstrate that the emergence of recessive genetic defects can be monitored by detecting de novo deleterious mutations in the genome of bulls used for artificial insemination. These results demonstrate the attractiveness of cattle as a model species in the post genomic era, particularly to confirm the genetic aetiology of isolated clinical case reports in humans.

KIT and melanoma predisposition in pigs: sequence variants and association analysis.

KIT mutations have been detected in different cancer subtypes, including melanoma. The gene also has been extensively studied in farm animals for its prominent role in coat color. The present work aimed at detecting KIT variants in a porcine model of cutaneous melanoma, the melanoblastoma-bearing Libechov Minipig (MeLiM). By sequencing exons and intron borders, 36 SNPs and one indel were identified. Of 10 coding SNPs, three were non-synonymous mutations, likely to affect the protein conformation. A promising variant, located in exon 19 (p.Val870Ala), was genotyped in a MeLiM × Duroc cross, and an association analysis was conducted on several melanoma-related traits. This variant showed a significant association with melanoma development, tumor ulceration and cutaneous invasion. In conclusion, although the KIT gene would not be a major causal gene for melanoma development in pig, its genetic variation could be influencing this trait.

Opposite regulation of tenascin-C and tenascin-X in MeLiM swine heritable cutaneous malignant melanoma.

Interactions between tumour cells and surrounding extracellular matrix (ECM) influence the growth of tumour cells and their ability to metastasise. It is thus interesting to compare ECM composition in tumours and healthy tissues. Using the recently described MeLiM miniature pig model of heritable cutaneous malignant melanoma, we studied the expression of two ECM glycoproteins, the tenascin-C (TN-C) and tenascin-X (TN-X), in normal skin and melanoma. Using semiquantitative RT-PCR, we observed a 3.6-fold mean increase of TN-C RNAs in melanoma compared to normal skin. Both stromal and tumour cells synthesise TN-C. On the contrary, TN-X RNAs decreased 30-fold on average in melanoma. This opposite regulation of TN-C and TN-X RNAs was confirmed at the protein level by indirect immunofluorescence. Whereas pig normal skin displayed a discrete TN-C signal at the dermo-epidermal junction, around blood vessels and hair bulbs, the swine tumour showed enhanced expression of TN-C in these areas and around stromal and tumour cells. In contrast, normal skin showed a strong TN-X staining at the dermo-epidermal junction and in the dermis, whereas this signal almost completely disappeared in the tumour. The results presented here describe a dramatic alteration of the ECM composition in swine malignant melanoma which might have a large influence on tumourigenesis or invasion and metastasis of melanoma cells.

Unlike tenascin-X, tenascin-C is highly up-regulated in pig cutaneous and underlying muscle tissue developing fibrosis after necrosis induced by very high-dose gamma radiation.

Fibrosis is characterized by proliferation of fibroblasts and deposition of extracellular matrix (ECM). As alterations in the composition of ECM may account for its chronic extension, we studied the expression of the tenascin-C (TN-C) and tenascin-X (TN-X) ECM glycoproteins in our pig model of the effects of accidental exposures to radiation, in which cutaneous and muscle fibrosis developed after the induction of necrosis after a high single dose (160 Gy at the skin surface) of gamma rays. We found that, in the healed fibrotic dermis and underlying muscle fibrosis, the amount of TN-C mRNA was increased up to 18- and 39-fold, respectively, compared to normal dermis, whereas the level of TN-X mRNA remained almost unchanged. In analyses by Western blotting, the two main TN-C isoforms of 235-240 and 190-200 kDa increased up to 45- and 105-fold in fibrotic tissues, respectively. The large isoform was expressed more strongly than the smaller, although in healed fibrotic scar tissues their ratio was lower in protein than in RNA. Compared to unirradiated skin, an immunohistological study revealed stronger TN-C staining at the dermo-epidermal junction and in areas of remodeling in healed skin. An intense extracellular staining was observed around myofibroblasts in muscle fibrosis. Therefore, the gene encoding TN-C is highly up-regulated in fibrotic tissues, and mechanisms regulating the levels of TN-C variants occur at both the RNA and protein levels. Each isoform might play a distinct role in the chronic activation of fibrosis by differentially regulating mechanisms like cell adhesion, migration or proliferation.

Coactivation of AP-1 activity and TGF-beta1 gene expression in the stress response of normal skin cells to ionizing radiation.

Activation of the AP-1 transcription factor and TGF-beta1 growth factor by ionizing radiation was studied both in vivo in pig skin, and in vitro in human fibroblasts and keratinocytes. Three and 6 h after irradiation, the Fos and Jun proteins and their binding activity to an AP-1 consensus sequence were strongly induced by high doses of gamma-rays. c-Fos, c-Jun and JunB proteins were found to be present in gel-shift complexes by probing with specific antibodies. Both keratinocytes and fibroblasts exhibited heightened AP-1 activity following irradiation. As we previously found that TGF-beta1 is involved in the development of skin lesions induced by radiation, TGF-beta1 gene expression was also examined. Two and 6 h after irradiation, the levels of TGF-beta1 transcripts were increased in skin. By immunostaining, TGF-beta1 protein levels were found to be increased in fibroblasts, keratinocytes and endothelial cells. As the TGF-beta1 promoter contains AP-1 binding sites, the relation between AP-1 activity and TGF-beta1 induction was addressed. The -365 TGF-beta1 promoter fragment, which contains a high affinity AP-1 site, exhibited increased binding to Jun and Fos proteins following irradiation. These results suggest that stress-inducible TGF-beta1 expression is mediated by the activation of AP-1 transcription factor.

Expression of 72-kDa gelatinase (MMP-2), collagenase (MMP-1), and tissue metalloproteinase inhibitor (TIMP) in primary pig skin fibroblast cultures derived from radiation-induced skin fibrosis.

In addition to producing matrix degradation for normal tissue remodeling and repair, matrix metalloproteinases (MMPs) are also involved in various pathologic processes. MMPs and the tissue inhibitor of MMPs (TIMP) were investigated in primary cultures of pig fibroblasts from radiation-induced dermal fibrosis and compared to normal dermal fibroblasts. The free gelatinolytic, collagenolytic, and caseinolytic activities secreted into the culture medium were evaluated against specific 3H denatured collagen type I, native helical collagen, and casein alpha, respectively. The 72- and 68-kilodalton (kDa) forms of type IV collagenase were investigated by protease zymography and quantified by semi-automated image analysis. Transcription of the interstitial collagenase (MMP-1) and TIMP genes was studied by Northern hybridization analysis. Results revealed that in fibrotic fibroblasts, the amount of MMP-1 mRNA was greatly reduced to undetectable levels whereas the amount of TIMP mRNA was increased fourfold compared to controls. Functional assays using specific 3H substrates demonstrated an overall decrease in free MMP activities. Concomitantly, catheptic collagenolytic activity decreased in fibrotic fibroblast extracts compared to controls. These results indicate that in addition to accumulating large amounts of collagen, proteoglycans, and fibronectin, pig fibroblasts from radiation-induced dermal fibrosis also promote connective tissue matrix formation by repressing MMP-1 and stimulating TIMP expression at the transcriptional level, and by reducing overall free MMP and catheptic collagenolytic activities at the post-transcriptional level. In contrast, enzymography assays and automated image analysis demonstrated no significant change in the 72-kDa type IV collagenase activity of fibrotic pig skin fibroblasts. This opposite regulation of 72-kDa collagenase type IV to that of MMP-1 seems to indicate that it has a specific role in remodeling the extracellular matrix during wound healing, fibrogenesis, and angiogenesis.

Preferential induction of c-fos versus c-jun protooncogene during the immediate early response of pig skin to gamma-rays.

The involvement of the nuclear protooncogenes c-fos and c-jun in the immediate early response of pig dermis cells was studied after in vivo gamma-irradiation. Following high radiation doses (8 to 48 Gy), the two protooncogenes were concomitantly induced, although c-fos induction was preferential. Both inductions were time and dose dependent. Therefore, the early response of the skin to high doses of radiation might involve heterodimeric activator protein 1 composed of c-Fos and c-Jun proteins. Following low radiation doses (0.5 to 2 Gy), c-jun was not induced. By contrast, dramatic c-fos induction was observed after 0.5 Gy, suggesting a specific role for c-fos at low doses.

Temporal modulation of TGF-beta 1 and beta-actin gene expression in pig skin and muscular fibrosis after ionizing radiation.

Although radiation-induced fibrosis has long been characterized by excess fibroblast proliferation and extracellular matrix deposition, the origin of cell activation in these complications of radiotherapy or radiation accidents is still controversial. The present work was designed to test the hypothesis that the abnormal production of TGF-beta 1 in irradiated tissues results in continuous signals for tissue repair and long-term cell activation. We examined gene expression of this growth factor in a well-characterized pig model of radiation-induced fibrosis, using Northern-blot and slot-blot hybridizations and indirect immunofluorescence. We found that the TGF-beta 1 mRNA level was increased 19-fold in the irradiated skin during the early erythematous phase, which started 3 weeks after irradiation. During the later phases of fibrosis, from 6 to 12 months after irradiation, the TGF-beta 1 gene was highly expressed in the repaired skin and the underlying muscular fibrotic tissue, with 10- and 8-fold maximal increases, respectively. In addition, we found that the beta-actin mRNA level was increased in the fibrotic tissue. Immunostaining for TGF-beta 1 revealed the presence of the protein in endothelial cells of capillaries, in myofibroblasts, and in the collagenous matrix of the fibrotic tissue. These results suggest that TGF-beta 1 may be one of the key cytokines involved in the cascade of events that leads to radiation-induced fibrosis, at both early and late stages.

Chromosomal anomalies in radiation-induced fibrosis in the pig.

R-banded karyotypes were established on fibroblasts from fibrotic tissues derived from experimental fibrosis induced in pigs, either surgically or by 64 Gy of gamma-rays from iridium-192. No chromosome aberrations were observed in the surgical fibrosis. In radiation-induced fibrosis, the high frequency of abnormal karyotypes and the frequent complexity of the chromosomal rearrangements suggest that the fibroblasts originated either from the 64-Gy area, or from the penumbra, but certainly not from non-irradiated areas. At early passages in vitro, almost all karyotypes were different, demonstrating a multiclonal origin of fibrotic tissue. At late passages (above 24), the situation was quite different, with the persistence of one or two clones only, demonstrating a strong selective pressure occurring in vitro.

Long-term overproduction of collagen in radiation-induced fibrosis.

Collagen metabolism was investigated in the fibrotic tissue which developed in pig thigh muscle 6 to 15 months after acute gamma irradiation. During this period, total collagen deposits in the fibrotic tissue increased 10-fold compared to the healthy muscle tissue. These deposits were composed mainly of type I and III collagen, and the type I/type III ratio was lower in the fibrotic than in the muscle tissue. Small pieces of both fibrotic and muscle tissue were incubated with [14C]proline. The [14C]hydroxyproline content of the fibrotic tissue reflected large concomitant increases in the synthesis of total collagen, mainly of types I and III, which rose 14- and 17-fold, respectively. Similarly, the level of type I and type III procollagen mRNAs rose 9- and 5-fold, respectively, in the fibrotic tissue versus the muscle tissue. These results suggest that procollagen gene transcription or RNA maturation in the cell nuclei is activated in the fibrotic tissue. The possibility that such activation is due to the long-term inflammatory state of this tissue is discussed.

Fibronectin and collagen gene expression during in vitro ageing of pig skin fibroblasts.

The fibronectin, collagen type I, and collagen type III genes code for three major proteins of the cell matrix. The age-related alterations in their expression were measured during the in vitro lifespan of pig skin fibroblasts. We observed changes in the transcription rate of these specific genes during ageing. The levels of fibronectin and type III collagen mRNA rose markedly during the senescence phase. The level of collagen type I mRNA decreased during cell ageing, while that of beta-actin did not change. As regards proteins, we observed a sharp increase in the secreted noncollagenous proteins and in the total proteins of the cell layer during senescence. On the contrary, the secretion of the collagenous proteins decreased during senescence. Moreover, most of the newly synthesized molecules of collagen were immediately degraded in the cells, before their extracellular secretion. The terminal phenotype of pig senescent cells was therefore characterized by overexpression of fibronectin and type III collagen genes and reduced expression of the type I collagen gene. Surprisingly, for fibronectin and type III collagen, that terminal phenotype resembled the one normally found in the fibroblasts during the processes of tissue repair, cicatrization, and development.