TP-40

To study inhibitory effect of recombinant transforming growth factor alpha-Pseudomonas exotoxin fusion protein (TP40) on proliferation of the human bladder cancer T24 cells.

Expression of epidermal growth factor receptor (EGFR) in cultured T24 cells was analyzed with Western blot assay. Human bladder cancer T24 cells were exposed to TP40 at 5 - 1 000 microg/L. Methyl thiazolyl tetrazolium assay was applied to evaluate the cell proliferation by measuring the absorbance (A) at 570 nm with a microplate reader. Tritium labeled thymine deoxyriboside ([(3)H]-TdR) uptake was measured to observe DNA synthesis. Competition assays were performed by the EGF at 1 - 7 500 microg/L.

Expression of EGFR was high in human bladder cancer T24 cells. Cell growth was suppressed by 10%, 19%, 27%, 41%, 47%, 53% and 61% after 96 h treatment with TP40 at 5, 50, 100, 250, 500, 750 and 1 000 microg/L, respectively. [(3)H]-TdR incorporation was 80%, 69%, 48% and 51% after 24 h, 48 h, 72 h, 96 h treatment with TP40 at 750 microg/L, respectively. When the concentration was 1 - 7 500 microg/L, EGF could block the inhibitory effect of TP40 to some extent.

Human bladder cancer T24 cells express EGFR at a high level. TP40 could inhibit the growth of T24 cells effectively in a dose- and time-dependent manner. The cytotoxic effects of TP40 were specifically mediated by EGFR.

The aim of this study was to investigate the potential role of foetal androgens in determining the sexual dimorphism in LH gene expression. Starting on day 30 p.c. pregnant sows were treated i.m. with testosterone propionate (TP) three times at 2-day intervals (TP30 treatment) or received additional TP treatment starting on day 40 p.c. (TP30/40). Sows were allowed to farrow and after frequent blood samples for LH determination were collected prepubertally (6 months) from the female offspring anterior pituitary LHbeta subunit mRNA levels were determined. In Experiment 2 pregnant sows were treated as TP30 before or received similar treatment starting on day 40 p.c. (TP40), but anterior pituitary LHbeta mRNA and plasma LH concentrations were determined at day 80 p.c. TP30 or TP30/40 treatment did not affect mean plasma LH concentrations nor LHbeta mRNA levels at 6 months of age but caused marked masculinization of external genitalia. At day 80 p.c. LHbeta mRNA and plasma LH levels were higher in female than in male foetuses. TP40 treatment suppressed LHbeta mRNA and plasma LH levels while TP30 treatment had no effect on LHbeta mRNA levels but caused masculinization of external genitalia in contrast to TP40. Our findings support the notion that the peak in plasma testosterone observed by others in the male pig foetus 5 weeks p.c. not only determines sexual differentiation of the LH surge mechanism but also LH gene expression in the foetus. The critical period for this process seems to succeed phenotypic differentiation (which appears to be largely completed before day 40 p.c.). The tonic mode of prepubertal LH gene expression and LH secretion in female pigs is not affected greatly by testosterone treatment at the stages of development that were investigated.