Abstract
We recently reported that rhabdomyosarcoma cell lines express and secrete interleukin 15 (IL-15), a tightly regulated cytokine with IL-2-like activity. To test whether the p53-impaired function that is frequently found in this tumour type could play a role in the IL-15 production, wild-type p53 gene was transduced in the human rhabdomyosarcoma cell line RD (which harbours a mutated p53 gene), and its effect on proliferation and expression of IL-15 was studied. Arrest of proliferation was induced by wild-type p53; increased proportions of G1-arrested cells and of apoptotic cells were observed. A marked down-modulation of IL-15 expression, at both the mRNA and protein level, was found in p53-transduced cells. Because a direct effect of IL-15 on normal muscle cells has been reported, the presence of IL-15 membrane receptors was studied by cytofluorometric analysis. Rhabdomyosarcoma cells showed IL-15 membrane receptors, which are down-modulated by wild-type p53 transfected gene. In conclusion, wild-type p53 transduction in human rhabdomyosarcoma cells induces the down-modulation of both IL-15 production and IL-15 receptor expression.
Highlights
We recently found that rhabdomyosarcoma cell lines express and secrete interleukin 15 (IL- 15) (Lollini et al 1997)
Transduction of p53 gene was perforned in the human rhabdomyosarcoma cell line RD. which is known to harbour a mutation in codon 248 of the p53 gene (Felix et al 1992)
Human rhabdomyosarcoma cells RD were wansduced with the tsp53 gene, whose product behaves like wild-type p53 at the pennissive temperature of 320C (Soddu et al, 1994). p53 expression level and transactivating action at both permissive and non-pennissive temperatures were determined on transfectant cells
Summary
Cells and p53 gene transductionTransduction of p53 gene was perforned in the human rhabdomyosarcoma cell line RD. which is known to harbour a mutation in codon 248 of the p53 gene (Felix et al 1992). Transduction of p53 gene was perforned in the human rhabdomyosarcoma cell line RD. Cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 370C in a humidified 7% carbon dioxide atmosphere. Carrying the temperature-sensitive p53 (ial-135) gene (ts-pS3) under the control of HaMSV-LTR and the selectable marker neo under the control of the RSV-LTR (Soddu et al 1994). Plasmid pRSVneo, carrying the selectable marker alone. 3 x 106 exponentially growing RD rhabdomyosarcoma cells were transfected by electroporation Transfectants were cloned by limiting dilution in medium containing 0.75 mg mll G418 (Gibco BRL): one clone designated RD-tsp and one control designated RD-Neo were studied. All transduced cells were routinely cultured at 37°C and shifted to the permissive temperature of 32°C for experimental studies
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