Interaction of SEWA sarcoma cell proteins with the intracisternal A-type particle long terminal repeat DNA sequence.

Intracisternal A-type particle (IAP) transcripts are endogenous retrovirus-like sequences expressed during specific stages of normal development and in a variety of murine tumors. In this study, we have analyzed two cell lines derived originally from the SEWA murine osteosarcoma and grown either as ascites or as solid tumors, for proteins that might regulate IAP expression. We found that subline AA7-NA, originally derived from the ascites tumor, expressed about five times more IAP RNA than the AS12-AD subline, which was derived from a solid tumor. In view of this finding, we examined the binding of cellular proteins from the two cell lines to the 5' end of an IAP long terminal repeat sequence. Gel retardation assays of DNA-protein complexes and DNase I footprinting assays identified several DNA sequences within the long terminal repeat fragment that were protected by protein extracts from both SEWA sublines. Gel retardation assays using specific synthetic oligonucleotide sequences that correspond to two of these protected regions revealed different patterns of DNA-protein complexes with extracts from the two SEWA sublines. These data suggest that expression of IAP sequences is regulated by complex mechanisms involving several proteins that appear to differ between the two sublines.

Ultraviolet light induces the expression of oncogenes in rat fibroblast and human keratinocyte cells.

The exposure of a polyoma virus transformed rat fibroblast cell line H3 to UV-C irradiation (254 nm) causes a transient increase in the abundance of RNAs for the cellular oncogenes c-H-ras, c-myc and c-fos, as well as RNAs homologous to an endogenous rat leukemia virus-related sequence (RaLV). Treatment with cycloheximide also causes a transient increase in the c-H-ras, c-myc and RaLV RNAs, with a time course similar to that obtained with UV irradiation. UV-C irradiation also causes a transient increase in the RNAs for c-H-ras and c-myc in an SV40 transformed human keratinocyte cell line SVK-14. Dose response studies with UV light at the various wavelengths found in sunlight indicate that UV-B (270-330 nm) and UV-A (345-440 nm) are much less potent than UV-C in inducing increased levels of c-H-ras and c-myc RNAs in SVK-14 cells. Thus, in addition to the well known mutagenic effects of UV irradiation, UV damage to DNA can also lead to increased expression of cellular oncogenes in both rodent fibroblasts and human keratinocytes.

Induction of asynchronous replication of polyoma DNA in rat cells by ultraviolet irradiation and the effects of various inhibitors.

The ability of various DNA damaging agents to induce asynchronous replication of polyoma DNA (APR) in rat cells carrying integrated copies of these DNA sequences may provide a useful model for understanding mechanisms of gene amplification. The present study has explored in detail the ability of UV irradiation to induce APR in the polyoma transformed rat fibroblast cell line H3. We have found that the optimum condition for induction of APR was obtained by irradiating the H3 cells with UV-C (wavelength, 254 nm) at 1-2 J/m2. Irradiation with UV-B (270-360 nm) was much less effective, and no induction of APR was obtained with even high doses of UV-A (345-440 nm). This action spectrum provides evidence that the critical target for induction of APR is DNA. We found that when normal rat fibroblasts were irradiated with UV-C and then fused to H3 cells, this also led to induction of APR. These results provide evidence that the induction of APR by UV-C is mediated by a trans-acting factor. The induction of APR by UV-C was inhibited by high doses of cycloheximide or actinomycin D, suggesting that the production of this trans-acting factor requires de novo protein and RNA synthesis. On the other hand, low doses of cycloheximide or actinomycin D alone were able to induce APR, perhaps by blocking the synthesis of cellular factors that normally inhibit APR. Thus, induction of APR by UV-C provides a useful system for identifying cellular factors that might mediate or prevent the asynchronous replication of various DNA sequences.

Comparative effects of aplysiatoxin, debromoaplysiatoxin, and teleocidin on receptor binding and phospholipid metabolism.

We have compared the activities of aplysiatoxin and debromoaplysiatoxin, two polyacetate marine algae toxins, with teleocidin, a tumor-promoting indole alkaloid from Streptomyces, with respect to inhibition of specific binding of epidermal growth factor, and phorbol-12,13-dibutyrate to their respective receptors and ability to stimulate the release of radioactivity from cells prelabeled with choline or arachidonic acid. Although these compounds have chemical structures that are quite different from the phorbol esters, both aplysiatoxin and teleocidin are essentially equipotent with the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate in all four assays. The fact that aplysiatoxin and teleocidin inhibit phorbol-12,13-dibutyrate-receptor binding suggests that their biological activities are mediated by binding to the same receptors utilized by the phorbol esters. Debromoaplysiatoxin, a debrominated form of aplysiatoxin, is about 10-fold weaker than aplysiatoxin in inhibiting epidermal growth factor and phorbol-12,13-dibutyrate-receptor binding, but is equipotent with aplysiatoxin in stimulating the release of lipid metabolites from the prelabeled cells. The results are discussed in terms of possible heterogeneity of cellular receptors for this group of compounds.

Molecular mechanisms of tumor promotion and multistage carcinogenesis.

Carcinogenesis is a multistep process resulting from a complex interaction between multiple factors, both environmental and exogenous. In contract to initiating agents that act by damaging cellular DNA, the primary targets of the phorbol ester tumor promoters are membrane-associated receptors. We have proposed a stereochemical model to explain the interaction of these amphiphilic molecules, and of teleocidin and aplysiatoxin, with this receptor system. The model is consistent with evidence that a complex between protein kinase C and phospholipid is the actual receptor for these compounds. Recent data we have obtained with a compound present in tung oil, 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate (HHPA), and twelve of its congener's (provided by Y. Ito et al.) are also consistent with our stereochemical model. We have studied phorbol ester receptors in a wide variety of tissue culture cell types. Our data, together with other findings, provide evidence for considerable receptor heterogeneity and this may relate to the pleiotropic effects of these compounds. We have found a case of "masked" receptors in a rat liver cell line and shown that it is due to a cell-associated esterase. Normal human melanocyte cultures contain phorbol ester receptors and this is of particular interest since these cells actually require these or related compounds for optimal growth (in collaboration with M. Eisinger). The receptor studies provide clues to how tumor promoters can, via inductive mechanisms, produce alterations in the structure and function of cell membranes. It is not known, however, how in the multistep carcinogenic process promoters enhance the eventual outgrowth of permanently altered tumor cells. We have found that TPA and teleocidin produce a marked enhancement of transformation of C3H 10T1/2 cells induced by transfection with h-ras human bladder cancer oncogene. These and other results are discussed in terms of the role of alterations in cellular oncogenes and transcriptional enhancer sequences during multistage carcinogenesis.

Anatomic restoration of congenital hip dysplasia in adulthood by total hip displacement.

Congenital dysplasia, treated or untreated, produces a hip joint difficult to reconstruct and is even more difficult when coxarthrosis supervenes producing significant disability. Total hip replacement can be dramatically successful in these patients, and equals those with coxarthrosis without congenital dislocation. The acetabulum must be totally reconstructed and relocated as near as possible to its original orientation. Usually a small straight stem femoral component must be placed into a generally constricted femoral canal. A thoughtful preoperative plan including X-ray templates is absolutely essential for a successful reconstruction without postoperative complications.