PTH-related peptide (
PTHrP) has been shown to be the major mediator of
hypercalcemia of
malignancy, but may also exert effects on cell growth and differentiation. The
Leydig cell tumor H-500, when implanted in Fischer rats, produces abundant
PTHrP and eventually causes the death of the host animal. In the present study we have used
antisense RNA technology to block the effects of
PTHrP in H-500 Leydig
tumor cells in vivo. The full-length rat
PTHrP complementary DNA encoding
amino acid -36-->141 was subcloned as an EcoRI-BglII insert in the antisense orientation into the mammalian expression vector pRc/CMV to produce the plasmid pRc-PAS. This plasmid was then stably transfected into the H-500 Leydig
tumor cells with a
Lipofectin reagent. After selection with the
neomycin derivative
G-418, a stable cell line, H-500-PTHrP-AS, was obtained which showed 80% inhibition of endogenous
PTHrP messenger RNA compared to wild-type or vector-only transfected H-500 cells.
Conditioned culture medium from these experimental cells showed a marked decrease in
PTHrP immunoreactivity and in the ability of the medium to stimulate
adenylate cyclase in UMR-106 rat
osteosarcoma cells. Furthermore, inhibition of
PTHrP production resulted in a significant increase in the doubling time of the H-500 cells. Transfection of the experimental plasmid into Rat-2 fibroblasts, which do not produce
PTHrP, had no effect on cell growth. Control and experimental cells were then implanted sc into male Fischer rats. Animals were killed at timed intervals, and their
tumor volumes were determined. Experimental animals receiving cells transfected with antisense
PTHrP plasmid showed near-normal levels of plasma
calcium and decreased expression of tumoral
PTHrP messenger RNA. These animals also showed a 30-70% lower
tumor volume during the course of the experiment compared to control animals. These studies have demonstrated that
PTHrP can play a role as a promoter of
tumor growth in vitro and in vivo.