Progressive abrogation of TGF‐β‐1 and EGF growth control is associated with tumour progression in <i>ras</i>‐transfected human keratinocytes

Game, Stephen; Huelsen, Andrea; Patel, Vyomesh; Donnelly, Mary; Yeudall, W. Andrew; Stone, A.; Fusenig, Norbert E.; Prime, Stephen S.

doi:10.1002/ijc.2910520322

Cited by 58 publications

(53 citation statements)

References 23 publications

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“…1079 TGF-,B1 production was increased and TGF-,B2 was decreased relative to HaCaT cells. These findings are consistent with the expression of TGF-fJ1 mRNA in Haras-transfected clones of HaCaT cells, but contrast with our previous data where loss of TGF-/ protein was noted by 11-3 and 11-4 cells (Game et al, 1992). In the present study, however, we measured immunogenic TGF-,B isoforms rather than total TGF-,B concentrated by ultrafiltration (Game et al, 1992).…”

Section: Discussionsupporting

confidence: 92%

“…These findings are consistent with the expression of TGF-fJ1 mRNA in Haras-transfected clones of HaCaT cells, but contrast with our previous data where loss of TGF-/ protein was noted by 11-3 and 11-4 cells (Game et al, 1992). In the present study, however, we measured immunogenic TGF-,B isoforms rather than total TGF-,B concentrated by ultrafiltration (Game et al, 1992). The data obtained for the Ha-ras-transfected HaCaT clones in the present study were supported by the findings in the only tumour-derived cell line containing mutant Ha-ras where TGF-#1 was markedly enhanced and TGF-fl2 undetectable in H357; the level of Ha-ras expression in this cell line is currently unknown.…”

Section: Discussionsupporting

confidence: 92%

“…The sandwich ELISA used in the present study facilitated the separate measurement of TGFp1, -,B2 and -,B3 in the same culture supernatant and, thereby, extended previous work by our group in which an assay of competitive inhibition of ligand binding was used to quantify total TGF-,B autocrine production (Game et al, 1992;Prime et al, 1994). The sensitivity and specificity of the sandwich ELISA in this study compared favourably with that described previously for TGF-,il (Danielpour, 1993).…”

Section: Discussionsupporting

confidence: 75%

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Dysregulation of autocrine TGF-beta isoform production and ligand responses in human tumour-derived and Ha-ras-transfected keratinocytes and fibroblasts

Fahey

Paterson²,

Stone³

et al. 1996

Br J Cancer

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Summary This study examined the autocrine production of TGF-,B1, -,B2 and -,B3 in culture supernatants from tumour-derived (H series, n =7; BICR series, n = 5), Ha-ras-transfected (n = 4) and normal (n =2) human keratinocytes using a sandwich enzyme-linked immunosorbent assay (ELISA). Detection limits were 39.0 pg ml-1' for TGF-,ll, 78.0 pg ml-' for TGF-fl2 and 1.9 ng ml-' for TGF-,B3. Tumour-derived oral keratinocytes predominantly produced less TGF-,B1 than normal oral epithelial cells; the expression of endogenous TGF-,B2 was variable. In keratinocytes containing mutant Ha-ras, TGF-,B1 production was enhanced and TGF-#2 was undetectable. TGF-,B3 mRNA was detected by reverse transcription -polymerase chain reaction (RT-PCR) but the protein was not detected in conditioned media, most probably because of the low detection limits of the ELISA for this isoform. Neutralisation experiments indicated that the latent TGF-,B peptide was secreted in keratinocyte conditioned medium. Seven tumour-derived keratinocyte cell lines (H series) and fibroblasts separated from normal (n = 1) and tumour-derived (n =2) keratinocyte cultures were examined for their response to exogenous TGF-fil, -fl2 and -fl3. Six of seven tumour-derived keratinocyte cell lines were inhibited by TGF-,B1 and TGF-,B2 (-,IB > -,2); one cell line was refractory to both TGF-fll and TGF-,B2. Keratinocytes were inhibited (4 of 7), stimulated (1 of 7) or failed to respond (2 of 7) to TGF-,B3. TGF-,Bl, -fl2 and -,B3 stimulated both normal and tumour-associated fibroblasts, but the tumour-associated fibroblasts showed less response to the ligands than their normal counterparts following prolonged treatment with each isoform. The results demonstrate variable autocrine production of TGF-,B isoforms by malignant keratinocytes, with loss of TGF-,lB generally associated with the tumour-derived phenotype and modification of endogenous isoform production dependent on the genetic background of the tumour cells. Further, the variable response of the tumour-derived keratinocytes and contiguous fibroblasts to the TGF-,B isoforms suggests that dysregulation of TGF-,B autocrine and paracrine networks are common characteristics of squamous epithelial malignancy.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 75%

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Dysregulation of autocrine TGF-beta isoform production and ligand responses in human tumour-derived and Ha-ras-transfected keratinocytes and fibroblasts

Fahey

Paterson²,

Stone³

et al. 1996

Br J Cancer

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“…One mg of each RNA sample was loaded on a 1% agarose gel and stained with ethidium bromide. A picture of the RNA gel is shown below the RNase protection assays (labeled RNA) wald and Green, 1977;Rubin et al, 1989;Game et al, 1992;H Smola, personal communication), our results indicate that stimulation of nucleotide biosynthesis via induction of enzymes with regulatory function could be a novel mechanism of growth factor action. Consistent with the inhibitory e ect of TGF-b and TNF-a on keratinocyte proliferation (Shipley et al, 1986;Detmar and Orfanos, 1990), these factors caused only a minor or no induction of the enzymes involved in nucleotide biosynthesis.…”

Section: Discussionmentioning

confidence: 70%

Growth factor – regulated expression of enzymes involved in nucleotide biosynthesis: a novel mechanism of growth factor action

1999

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Keratinocyte growth factor (KGF) is a potent and speci®c mitogen for epithelial cells, including the keratinocytes of the skin. We investigated the mechanisms of action of KGF by searching for genes which are regulated by this growth factor in cultured human keratinocytes. Using the di erential display RT ± PCR technology we identi®ed the gene encoding adenylosuccinate lyase [EC 4.3 [EC 3.5.2.3]). Expression of all of these enzymes was upregulated after treatment with KGF and also with epidermal growth factor (EGF), indicating that these mitogens stimulate nucleotide production by induction of these enzymes. To determine a possible in vivo correlation between the expression of KGF, EGF and the enzymes mentioned above, we analysed the expression of the enzymes during cutaneous wound repair, where high levels of these mitogens are present. Indeed, we found a strong mRNA expression of all of these enzymes in the EGF-and KGF-responsive keratinocytes of the hyperproliferative epithelium at the wound edge, indicating that their expression might also be regulated by growth factors during wound healing.

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“…TGF-␤ is an important negative regulator of epithelial cell proliferation, the antiproliferative response to which may become diminished during squamous cell carcinogenesis. 39,40 In addition to its contribution to epithelial homeostasis and development, TGF-␤ plays a role in tumor invasion and metastasis. [41][42][43] Conceivably, this might occur through modulation of laminin-5 expression, though we have not tested this hypothesis directly.…”

Section: Discussionmentioning

confidence: 99%

Laminin‐γ2 overexpression in head‐and‐neck squamous cell carcinoma

Patel

Aldridge

Ensley

et al. 2002

Intl Journal of Cancer

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To identify molecular markers for the progression of headand-neck squamous cell carcinoma (HNSCC), we used RNA arbitrarily primed (RAP) PCR to determine the qualitative and quantitative differences in gene expression between normal epithelial cells, those derived from dysplastic oral mucosa and invasive and metastatic HNSCC. Three differentially expressed DNA fragments (RAP20, RAP21, RAP26) that were upregulated in a tumor cell line (T45) were identified as being regions of the ␥2 subunit of human laminin-5. Northern blot analysis of total cellular RNA revealed overexpression of these transcripts in 6 of 7 HNSCC cell lines compared with normal epidermal keratinocytes. In contrast, no differences were observed in HeLa (cervical carcinoma) or HCT116 (colon carcinoma) cells. Laminin is a major component of the basement membrane, which performs multiple biologic roles, including cell attachment, spreading and migration as well as being involved in cellular proliferation and differentiation. 1,2 Structurally, laminin is a heterotrimeric protein comprising ␣, ␤ and ␥ subunits, of which several variants exist. 3 Thus, different subunit combinations result in a family of laminins. 3,4 Specific biologic activities of different laminins may be reflected in the fact that these molecules show tissue-specific expression. For example, laminin-5 is considered to be an epithelial laminin as it is found as a component of basement membranes in epithelial tissues but not in mesenchyme. 5,6 Furthermore, cytokines that regulate epithelial homeostasis, such as transforming growth factor (TGF)-␤, modulate the expression of laminin-5 polypeptides in epidermal keratinocytes. 7 A role for laminin isoforms in tumor cell invasion is becoming apparent. 8 For instance, attachment of cancer cells is mediated in part by laminins. 9,10 Additionally, increased cell-surface expression of laminin was found in highly metastatic fibrosarcoma cells compared to cells of low metastatic potential. 11 Furthermore, expression and activity of matrix metalloproteases, which play critical roles in cellular invasion, are elevated in the presence of laminin, 12 and metastasis of melanoma cells is also enhanced. 13 Further studies have demonstrated that attachment and metastasis of tumor cells can be inhibited by incubation with antilaminin antibodies 14,15 or synthetic laminin peptides. 16 Several reports have implicated laminin-5 in epithelial tumor cell invasion. In one study, overexpression of the ␥2 chain of laminin-5 was found in 26 of 27 carcinomas, while various mesenchymal tumors did not express this molecule. 17 Furthermore, the highest expression of ␥2 occurred at areas of active invasion in colon adenocarcinomas, and a clear correlation was found with tumor budding. 17 In a subsequent study, these findings were extended to examine expression in a larger series of human cancers, 18 demonstrating that levels of ␥2 were indeed the highest in squamous carcinomas, particularly in cells adjacent to surrounding stromal tissue and in deeply invading cell ne...

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Progressive abrogation of TGF‐β‐1 and EGF growth control is associated with tumour progression in ras‐transfected human keratinocytes

Cited by 58 publications

References 23 publications

Dysregulation of autocrine TGF-beta isoform production and ligand responses in human tumour-derived and Ha-ras-transfected keratinocytes and fibroblasts

Dysregulation of autocrine TGF-beta isoform production and ligand responses in human tumour-derived and Ha-ras-transfected keratinocytes and fibroblasts

Growth factor – regulated expression of enzymes involved in nucleotide biosynthesis: a novel mechanism of growth factor action

Laminin‐γ2 overexpression in head‐and‐neck squamous cell carcinoma

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