Production of epidermal growth factor in human prostatic cells cultured in vitro.
The Epidermal Growth Factor (EGF) plays an important role in the regulation of in vitro growth of prostate cells inducing a strong mitogenic effect. Nevertheless in our previous study we observed that the treatment of human hypertrophic prostate cell line U285 with exogenous EGF produces a restricted effect on the cellular growth rate. This phenomenon could be due to the capacity of the cells to produce EGF. In this study we aimed to verify this hypothesis by evaluating the presence of mRNA of EGF and EGF receptor (EGF-R) and of their translation products in U285 cells, before and after the treatment with suramin and exogenous EGF. Moreover we studied the effects exerted by these substances on the proliferative rate of the cells U285 after different treatment protocols. The presence in the cells of mRNA for EGF and EGF-R and of their translation products was demonstrated by means of reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemical methods respectively. The modification of growth rate induced by these drugs was studied by FRAME Cytotoxicity Test. The operative modalities adopted to carry out these growth assays tended to 1) focus the effects of suramin in relation to in vitro cellular growth phase; 2) verify the reversibility of its effects; 3) ascertain if it was possible to antagonize the action of suramin by adding exogenous EGF. The results obtained from the RT-PCR showed the presence, in the control cells and in the treated ones, of mRNA coding for EGF and EGF-R. The immunocytochemical analysis indicated that 20% of the control cells are EGF positive, and 83% are EGF-R positive, confirming the results obtained with RT-PCR. Moreover, these stainings showed that the treatment with EGF does not significantly modify the percentage of cells marked by the anti-EGF antibody, while treatments with suramin and suramin plus EGF double this percentage. None of the treatments modifies the percentage of EGF-R positive cells. The growth assays showed that the exposition to highest doses of suramin in the first 24 h of cultures causes a decrease (p < 0.05) of the cellular proliferation during the following 48 h and 72 h and that these effects are irreversible. Moreover, a contemporaneous exposition of the cells to EGF and suramin at seeding strengthens the cytotoxic action of the last drug. To sum up, the demonstration of the presence in the U285 cells of mRNA coding for EGF and EGF-R and of the corresponding proteins, confirms the hypothesis that these cells can produce EGF. Moreover, the cytotoxicity experiments allowed a focusing of the role of the endogenous EGF in the regulation of the U285 cells proliferation and confirmed the importance of biological events that take place in U285 cells during the first 24 h of culture.
De Angeli
S
S
Cell Culture Laboratory of the Transfusion Center, Hospital of Treviso, Italy.
Buoro
S
S
Fandella
A
A
Anselmo
G
G
Palù
G
G
Mingrino
R
R
Parnigotto
P P
PP
D000704:Analysis of Variance; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D004815:Epidermal Growth Factor / Q000032:analysis / Q000235:genetics* / Q000494:pharmacology*; D066246:ErbB Receptors / Q000032:analysis / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D011467:Prostate / Q000378:metabolism*; D011470:Prostatic Hyperplasia; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013498:Suramin / Q000633:toxicity*
D012333:RNA, Messenger; D013498:Suramin; D004815:Epidermal Growth Factor; D066246:ErbB Receptors