p53 gene structure and chromosome 17p alleles were studied in the three human prostate cancer cell lines, LNCaP, DU-145, and PC-3. Our laboratory has two separate culture lines of the LNCaP human prostate cancer cells. One strain, LNCaP-GW, had a mutation in one of two alleles at position 273 (arg > his). This mutation could not be detected in a second strain of LNCaP, LNCaP-ATCC. Immunohistochemical staining for P53 protein in the cell lines indicated that protein overexpression in LNCaP was heterogeneous, even in clonal isolates derived from LNCaP-GW that contained the codon 273 mutation in every cell. We also performed in vitro and in vivo growth analysis to compare the LNCaP-GW and LNCaP-ATCC cells. LNCaP-GW grew more rapidly than LNCaP-ATCC in vitro. However, LNCaP-ATCC formed tumors efficiently when inoculated into nude mice, whereas LNCaP-GW formed tumors much less efficiently. Consideration must be given to the notion that some of these p53 mutations arose during in vitro passage. We also confirmed published findings with two other human prostate cancer cell lines. In DU-145, two mutations were found in the p53 gene. A mutation at codon 274 (pro > leu) and a second mutation at codon 223 (val > phe) were present. PC-3 cells were hemizygous for chromosome 17p. The single copy of the p53 gene had a base pair deletion at codon 138 that generated a frame shift and a new in-frame stop codon at position 169.

The biological activity of transforming growth factor beta1 (TGF-beta) is controlled by its secretion as a latent complex in which it is noncovalently associated with latency-associated peptide (LAP). Activation is the extracellular process in which TGF-beta is released from LAP, and is considered to be a primary regulatory control. We recently reported rapid and persistent changes in TGF-beta immunoreactivity in conjunction with extracellular matrix remodeling in gamma-irradiated mouse mammary gland. Our hypothesis is that these specific changes in immunoreactivity are indicative of latent TGF-beta activation. In the present study, we determined the radiation dose response and tested whether a functional relationship exists between radiation-induced TGF-beta and collagen type III remodeling. After radiation exposures as low as 0.1 Gy, we detected increased TGF-beta immunoreactivity in the mammary epithelium concomitant with decreased LAP immunostaining, which are events consistent with activation. Quantitative image analysis demonstrated a significant (P=0.0005) response at 0.1 Gy without an apparent threshold and a linear dose response to 5 Gy. However, in the adipose stroma, loss of LAP demonstrated a qualitative threshold at 0.5 Gy. Loss of LAP paralleled induction of collagen III immunoreactivity in this tissue compartment. We tested whether TGF-beta mediates collagen III expression by treating animals with TGF-beta panspecific monoclonal antibody, 1D11.16, administered i.p. shortly before irradiation. Radiation-induced collagen III staining in the adipose stroma was blocked in an antibody dose-dependent manner, which persisted through 7 days postirradiation. RNase protection assay revealed that radiation-induced elevation of total gland collagen III mRNA was also blocked by neutralizing antibody treatment. These data provide functional confirmation of the hypothesis that radiation exposure leads to latent TGF-beta activation, support our interpretation of the reciprocal shift in immunoreactivity as evidence of activation, and implicate TGF-beta as a mediator of tissue response to ionizing radiation. The sensitivity of activation to low radiation doses points to a potential role for TGF-beta in orchestrating tissue response to oxidative stress. As such, radiation may be useful as a probe to delineate the consequences of latent TGF-beta activation in situ.

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