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Release of transforming growth factor beta by M9 fibrosarcoma
Cell Biology International
Reports, Vol. 14, Abstracts Supplement
ENDOGENOUS IGFs MODULATES THE MITOGENIC RESPONSEOF RAT THYOCYTESIN COLTURE Giovanni Vlllone, Bianca M Venazlani. Franccsca De Amicis, Rodolfo Frunzio, Donatella Tramontano. Dpt. Universiti di Reggio di Medlcina Sperimentale, Calabria, Cataniaro; CEOS/CNRand Dpt. di Biologic e Patologia Cellulare e Molecolare, Universitl di NapoUT Naples, Italy. PRTLS cells have been transfected with the sequence coding for rat IGF-II under the control of along with a dominant promoter a retroviral selectable marker. Northern blot analysis of total RNA showed the presence of a mRNAspecific for the IGF-II In most of the neomiclne transfected resistent clones. Clones TR7 and TR12 expressed intermediate and high levels of mRNAfor transfected IGF-II. They possibly secreted into the culture media a peptlde immunologically related 30 IGF-II as H-thymidine demonstrated by the high level of incorporation in basal conditions. compared to FRTLS. In addition sm1.2, a monoclonal antibody significantly inhibits basal ggainst rat IGF-II, these Ii-thymidine incorporation in cells. Consistent with the presence of an IGF-related pepelde in the culture media are the lack of response to tha mltogenic action of IGFs or insulin, 794 the mitogenic effect of adenosine. Binding of I-IGF to Tr7 and Tr12 is significantly reduced in respect to that observed in FRTL5 cells, although the affinity of the receptors is similar in the two TSH stimulate DNA synthesis in groups of cells. clones Tr7 and Tr12 as in the parental FRTLS. Finally, media conditioned by TR7 and TR12 increase basal thymidine incorporation in FRTLS cells and amplify the mitogenic action of TSH. Endogenous IGFs cell proliferation by thyroid regulate -Y modulating the mitogenic effect of TSH and by supporting their TSH-Independent. growth.
P49
P51
RELEASE OF TRANSFORMING GROWTH FACTORBJ!TA BY M9 F’IkROSARCOMA.
Vilma Giandomenico, Monica Rapino, and Andreina Poggi. Laboratory of Vascular and Tumour Cell Biology, Consorzio Mario Negri Sud, 66030 S. Maria Imbaro, Italy. Transforming growth factor beta (TGFB) is released by several tumor cell lines in cultue and plays a role on cell growth control and cell matrix deposition. We measured the production of TGFB from cultured cells derived by two murine fibrosanzoma lines with different metastatic potential, M4 and M9. TGFB-like activity was measured as the ability to inhibit serum-sdmulated 3H-thymidine incorporation by rat tyroid cells FRTL-5 and by mink epithelial cells MvLu. We found that tumour cell extracts of both cell lines were devoid of activity, whereas the conditioned media (CM) showed comparable inhibitory activity; 50% inhibition was obtained at the dose of 20 @ml. The activity of CM was only partially inhibited by incubation with specific anti-TGFB antibody. CM’s from both cell lines were able to displace lzI-TGFB binding to MvLu cells and to stimulate growth of NRK cells in soft agar. CM from M9 cells was partially purified by chromatography on Bio Gel P60 in 1M acetic acid The effect of human TGFB, on the adhesion of M9 tumour cells to fibronectin and other substrata was also measured, in order to evaluate the role of this peptide on tumour development. Partially supported by the Italian Association for Cancer Research and by Italian CNR (Project n. 89.01270.04).
1990
91
MITOGENIC PROPERTIES OF CONDITIONED MEDIA FROM K-ras TRANSFORMED EPITHELIAL CELLS. Paola Cucchi, Salvatore Valitutti, Daniela Corda. lstituto di Ricerche Farmacologiche “Mario Negri”, Consorzio Mario Negri Sud, 66030 S. M. lmbaro (Chieti), Italy. Conditioned media from K-ras transformed cells (KiKi and A6 cell lines) derived from non tumoral thyroid cells (FRTW), have been found to exert a mitogenic activity on the parent FRTLS line. FRTLS cells mantain markers of differentiation: they are able to synthesize thyroglobulin and their growth is dependent on thyrotropin. When transformed by the K-ras oncogene these cells loose their differentiated properties, acquire a malignant phenotype and grow in a thyrotropin independent manner (KiKi and A6 cell lines). Conditioned media from KiKi and A6 cell cultured in serum free conditions, were able to stimulate [3 HI-thymidine (Tdr) uptake in the normal FRTL-5 cells. This effect was dose dependent and occurred both in the presence (300% increase in Tdr incorporation) and in the absence (IOO-200% increase in Tdr inorporation) of serum. The mitogenic factor present in the transformed cell medium was heat stable, since the mitogenic activity was unaffected by a 10 min incubation at 50” C or a 3 min incubation at 100” C. Preliminary observations performed with partially purified materials suggest that the mitogenic factor has a low molecular weight (~20 kDa). We have tested the activity of known heat stable growth factors (EGF, PDGF, TGFa) on FRTL-5 cells and found no detectable increase in Tdr uptake, even at concentrations up to twenty fold higher than those known to be mitogenic for fibroblast cultures. Our data suggest that an epithelial growth factor may be produced by K-ras transformed cells.
P50
*This study was supported by A.I.R.C., Fidia SpA. CasMez (PS.35.93OND).
METASTATIC PHENOTYPE: GRovlg FACfOR HEIWNCE ANDIM’EGRINXPESSION Giulio Tibursi, M. Pia Gentileschi ,RitaF&ioni, Letkia Chino, Danilo hrretti, Adr SIcchi - Inb,Oncoamesi Mokcoluekt.. Regina Elena- 00161Roma ITALIA P52
Since the ymth of malignant cells at secondarysites coulddspmd on their ability to mapondto mhwnvironmenb. and the adhesionof cells to substratels med&d by molecuIe8of the rstrscellukr mat&, the #roti factor dependmce and the l xpressien of estracellulrr matrix recrptors(int&nr) of 3LL.metubuc vudmts have beenstudied.The &l&y of the varkt liner to grm in monolryet md in soft yu cultures, ln the plwsnce or ia the abancr of different lrorch factors or swum, has bwn an8ly&d md correlated rith their m&at&c potential. Resultssported kmonstrate that tumor crlls exprssring higher metuhtic potenti& Jn exhibit highor canacitvto RIVVmd orolifentr in alI the culture coiditioni t&d, -in&pmitmtIy on the addition of rxo~enous growth fwtors or serumAnalysis of TGF-Bl mRNA expressionon vwht lines suer&a & p&tern of uitocrins m for 3LLm&st8tic cells.?dorwerresults also demonstrateth8t a nwel integrln alpha6/54 is specifically rxpresssd on highly metuktic 3U cells, ihich i? +enf on lwer meta&$zin~ ones.Ity contrast, ~~~rn;ceptor(alpha6/B1 1 w hl6hly expressedon In conclusion ‘datapresenteddemonstmte that 3LL.cells endoved rlth highor m&static potenUrl M more independenton the microenvironmentel conditiear than the her mautrrhina ones, have urtucrine e&p&city, and express sign&ant amount of a novel integrin, dsflned asalpha6/84. Partidy suppwtedby A.I.R.C.and C.N.R.
Reports, Vol. 14, Abstracts Supplement
ENDOGENOUS IGFs MODULATES THE MITOGENIC RESPONSEOF RAT THYOCYTESIN COLTURE Giovanni Vlllone, Bianca M Venazlani. Franccsca De Amicis, Rodolfo Frunzio, Donatella Tramontano. Dpt. Universiti di Reggio di Medlcina Sperimentale, Calabria, Cataniaro; CEOS/CNRand Dpt. di Biologic e Patologia Cellulare e Molecolare, Universitl di NapoUT Naples, Italy. PRTLS cells have been transfected with the sequence coding for rat IGF-II under the control of along with a dominant promoter a retroviral selectable marker. Northern blot analysis of total RNA showed the presence of a mRNAspecific for the IGF-II In most of the neomiclne transfected resistent clones. Clones TR7 and TR12 expressed intermediate and high levels of mRNAfor transfected IGF-II. They possibly secreted into the culture media a peptlde immunologically related 30 IGF-II as H-thymidine demonstrated by the high level of incorporation in basal conditions. compared to FRTLS. In addition sm1.2, a monoclonal antibody significantly inhibits basal ggainst rat IGF-II, these Ii-thymidine incorporation in cells. Consistent with the presence of an IGF-related pepelde in the culture media are the lack of response to tha mltogenic action of IGFs or insulin, 794 the mitogenic effect of adenosine. Binding of I-IGF to Tr7 and Tr12 is significantly reduced in respect to that observed in FRTL5 cells, although the affinity of the receptors is similar in the two TSH stimulate DNA synthesis in groups of cells. clones Tr7 and Tr12 as in the parental FRTLS. Finally, media conditioned by TR7 and TR12 increase basal thymidine incorporation in FRTLS cells and amplify the mitogenic action of TSH. Endogenous IGFs cell proliferation by thyroid regulate -Y modulating the mitogenic effect of TSH and by supporting their TSH-Independent. growth.
P49
P51
RELEASE OF TRANSFORMING GROWTH FACTORBJ!TA BY M9 F’IkROSARCOMA.
Vilma Giandomenico, Monica Rapino, and Andreina Poggi. Laboratory of Vascular and Tumour Cell Biology, Consorzio Mario Negri Sud, 66030 S. Maria Imbaro, Italy. Transforming growth factor beta (TGFB) is released by several tumor cell lines in cultue and plays a role on cell growth control and cell matrix deposition. We measured the production of TGFB from cultured cells derived by two murine fibrosanzoma lines with different metastatic potential, M4 and M9. TGFB-like activity was measured as the ability to inhibit serum-sdmulated 3H-thymidine incorporation by rat tyroid cells FRTL-5 and by mink epithelial cells MvLu. We found that tumour cell extracts of both cell lines were devoid of activity, whereas the conditioned media (CM) showed comparable inhibitory activity; 50% inhibition was obtained at the dose of 20 @ml. The activity of CM was only partially inhibited by incubation with specific anti-TGFB antibody. CM’s from both cell lines were able to displace lzI-TGFB binding to MvLu cells and to stimulate growth of NRK cells in soft agar. CM from M9 cells was partially purified by chromatography on Bio Gel P60 in 1M acetic acid The effect of human TGFB, on the adhesion of M9 tumour cells to fibronectin and other substrata was also measured, in order to evaluate the role of this peptide on tumour development. Partially supported by the Italian Association for Cancer Research and by Italian CNR (Project n. 89.01270.04).
1990
91
MITOGENIC PROPERTIES OF CONDITIONED MEDIA FROM K-ras TRANSFORMED EPITHELIAL CELLS. Paola Cucchi, Salvatore Valitutti, Daniela Corda. lstituto di Ricerche Farmacologiche “Mario Negri”, Consorzio Mario Negri Sud, 66030 S. M. lmbaro (Chieti), Italy. Conditioned media from K-ras transformed cells (KiKi and A6 cell lines) derived from non tumoral thyroid cells (FRTW), have been found to exert a mitogenic activity on the parent FRTLS line. FRTLS cells mantain markers of differentiation: they are able to synthesize thyroglobulin and their growth is dependent on thyrotropin. When transformed by the K-ras oncogene these cells loose their differentiated properties, acquire a malignant phenotype and grow in a thyrotropin independent manner (KiKi and A6 cell lines). Conditioned media from KiKi and A6 cell cultured in serum free conditions, were able to stimulate [3 HI-thymidine (Tdr) uptake in the normal FRTL-5 cells. This effect was dose dependent and occurred both in the presence (300% increase in Tdr incorporation) and in the absence (IOO-200% increase in Tdr inorporation) of serum. The mitogenic factor present in the transformed cell medium was heat stable, since the mitogenic activity was unaffected by a 10 min incubation at 50” C or a 3 min incubation at 100” C. Preliminary observations performed with partially purified materials suggest that the mitogenic factor has a low molecular weight (~20 kDa). We have tested the activity of known heat stable growth factors (EGF, PDGF, TGFa) on FRTL-5 cells and found no detectable increase in Tdr uptake, even at concentrations up to twenty fold higher than those known to be mitogenic for fibroblast cultures. Our data suggest that an epithelial growth factor may be produced by K-ras transformed cells.
P50
*This study was supported by A.I.R.C., Fidia SpA. CasMez (PS.35.93OND).
METASTATIC PHENOTYPE: GRovlg FACfOR HEIWNCE ANDIM’EGRINXPESSION Giulio Tibursi, M. Pia Gentileschi ,RitaF&ioni, Letkia Chino, Danilo hrretti, Adr SIcchi - Inb,Oncoamesi Mokcoluekt.. Regina Elena- 00161Roma ITALIA P52
Since the ymth of malignant cells at secondarysites coulddspmd on their ability to mapondto mhwnvironmenb. and the adhesionof cells to substratels med&d by molecuIe8of the rstrscellukr mat&, the #roti factor dependmce and the l xpressien of estracellulrr matrix recrptors(int&nr) of 3LL.metubuc vudmts have beenstudied.The &l&y of the varkt liner to grm in monolryet md in soft yu cultures, ln the plwsnce or ia the abancr of different lrorch factors or swum, has bwn an8ly&d md correlated rith their m&at&c potential. Resultssported kmonstrate that tumor crlls exprssring higher metuhtic potenti& Jn exhibit highor canacitvto RIVVmd orolifentr in alI the culture coiditioni t&d, -in&pmitmtIy on the addition of rxo~enous growth fwtors or serumAnalysis of TGF-Bl mRNA expressionon vwht lines suer&a & p&tern of uitocrins m for 3LLm&st8tic cells.?dorwerresults also demonstrateth8t a nwel integrln alpha6/54 is specifically rxpresssd on highly metuktic 3U cells, ihich i? +enf on lwer meta&$zin~ ones.Ity contrast, ~~~rn;ceptor(alpha6/B1 1 w hl6hly expressedon In conclusion ‘datapresenteddemonstmte that 3LL.cells endoved rlth highor m&static potenUrl M more independenton the microenvironmentel conditiear than the her mautrrhina ones, have urtucrine e&p&city, and express sign&ant amount of a novel integrin, dsflned asalpha6/84. Partidy suppwtedby A.I.R.C.and C.N.R.