Unique recombinant human ribonuclease and inhibition of Kaposi's sarcoma cell growth.
BACKGROUND
Preparations of human chorionic gonadotropin (hCG) have been shown to exhibit anti-Kaposi's sarcoma (KS) activity, but the identity of the responsible agent(s) remains controversial. One candidate agent is an eosinophil-derived neurotoxin (EDN)-like polypeptide that contaminates preparations of hCG. We have genetically engineered a unique form of hEDN, which is a ribonuclease, and have evaluated the cytotoxic effects of the recombinant 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, corresponding to the proximal part of the hEDN signal peptide (serine, leucine, histidine, and valine; positions -4 to -1, respectively), by use of the polymerase chain reaction and an hEDN complementary DNA. The recombinant protein was isolated from bacterial inclusion bodies. The cytotoxic activity of this hEDN variant, (-4)rhEDN, was tested on KS Y-1 cells and human glioma, melanoma, breast carcinoma, and renal carcinoma cells.


RESULTS
Approximately half of the anti-KS activity in a crude commercial preparation of hCG was associated with a polypeptide that reacted with anti-recombinant-hEDN (rhEDN) polyclonal antibodies. Although rhEDN protein displayed little cytotoxicity against KS Y-1 cells (IC50 [50% inhibition concentration] = >100 microg/mL), (-4)rhEDN markedly inhibited cell viability (IC50 = 6 microg/mL). Neither version of rhEDN inhibited the viability of other tested human cancer cell types.


CONCLUSIONS
A four amino acid extension of the amino-terminus of rhEDN confers cytotoxicity against KS Y-1 cells in vitro. Design of the (-4)rhEDN 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 responsible for anti-KS activity. A purified molecule is thus available for in vitro and in vivo mechanistic and, possibly, future clinical studies.
Newton
D L
DL
Intramural Research Support Program, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, MD 21702-1201, USA.
Rybak
S M
SM
D000970:Antineoplastic Agents / Q000627:therapeutic use*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy; D018076:DNA, Complementary / Q000138:chemical synthesis; D047208:Eosinophil-Derived Neurotoxin; D005813:Genes, Synthetic; D006639:Histidine / Q000235:genetics; D006801:Humans; D007680:Kidney Neoplasms / Q000188:drug therapy; D007930:Leucine / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011506:Proteins / Q000627:therapeutic use*; D011994:Recombinant Proteins / Q000627:therapeutic use; D012259:Ribonuclease, Pancreatic / Q000627:therapeutic use*; D012260:Ribonucleases; D012514:Sarcoma, Kaposi / Q000188:drug therapy* / Q000378:metabolism; D012694:Serine / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014633:Valine / Q000235:genetics
D000970:Antineoplastic Agents; D018076:DNA, Complementary; D011506:Proteins; D011994:Recombinant Proteins; D012694:Serine; D006639:Histidine; D047208:Eosinophil-Derived Neurotoxin; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D007930:Leucine; D014633:Valine