Unique recombinant human ribonuclease and inhibition of Kaposi's sarcoma cell growth

Background: Preparations of human chorionic gonadotropin (hCG) have been sh own to exhibit anti-Kaposi's sarcoma (KS) activity, but the identity of the responsible agent(s) remains controversial. One candidate agent is an eosi nophil-derived neurotoxin (EDN)-like polypeptide that contaminates preparat ions of hCG, We have genetically engineered a unique form of hEDN, which is a ribonuclease, and have evaluated the cytotoxic effects of the recombinan t protein on KS Y-1 cells and on cells of other cancer types. Methods: The amino-terminus of hEDN was extended by four amino acid residues, correspond ing to the proximal part of the hEDN signal peptide (serine, leucine, histi dine, and valine; positions -4 to -1, respectively), by use of the polymera se chain reaction and an hEDN complementary DNA, The recombinant protein wa s isolated from bacterial inclusion bodies. The cytotoxic activity of this hEDN variant, (-4)rhEDN, was tested on KS Y-1 cells and human glioma, melan oma, breast carcinoma, and renal carcinoma cells. Results: Approximately ha lf of the anti-KS activity in a crude commercial preparation of hCG was ass ociated with a polypeptide that reacted with anti-recombinant-hEDN (rhEDN) polyclonal antibodies, Although rhEDN protein displayed little cytotoxicity against KS Y-l cells (IC50 [50% inhibition concentration] = >100 mu g/mL), (-4)rhEDN markedly inhibited cell viability (IC50 = 6 mu g/mL). Neither ve rsion of rhEDN inhibited the viability of other tested human cancer cell ty pes. Conclusions: A four amino acid extension of the amino-terminus of rhED N confers cytotoxicity against KS Y-1 cells in vitro, Design of the (-4)rhE DN variant was based on the sequence of a natural human protein associated with hCG, Our results suggest that (-4)rhEDN is one of the agents in hCG re sponsible for anti-KS activity. A purified molecule is thus available for i n vitro and in vivo mechanistic and, possibly, future clinical studies.