A dimeric mutant of human pancreatic ribonuclease with selective cytotoxicity toward malignant cells

Abstract

Monomeric human pancreatic RNase, devoid of any biological activity other than its RNA degrading ability, was engineered into a dimeric protein with a cytotoxic action on mouse and human tumor cells, but lacking any appreciable toxicity on mouse and human normal cells. This dimeric variant of human pancreas RNase selectively sensitizes to apoptotic death cells derived from a human thyroid tumor. Because of its selectivity for tumor cells, and because of its human origin, this protein represents a potentially very attractive, novel tool for anticancer therapy.

Figure 1

Comparison of the amino acid sequence of human pancreas RNase (HP-RNase) with that of bovine seminal RNase (BS-RNase). For the latter, only the differences are indicated. Evidenced with a black background are the four BS-RNase residues grafted in the HP-RNase sequence to construct the dimeric mutant.

Figure 2

SDS/PAGE electrophoresis on a 15% polyacrylamide gel of the monomeric and dimeric HP-RNase variants. Lanes: 1, dimeric BS-RNase and monomeric RNase A; 2, monomeric LCCK-HP-RNase; 3 and 4, dimeric HHP-RNase, analyzed under nonreducing (lane 3) and reducing (lane 4) conditions.

Figure 3

Effects of dimeric HHP-RNase on murine fibroblasts. Malignant SVT2 fibroblasts (closed symbols) or normal 3T3 fibroblasts (open symbols) were grown in the absence or in the presence of HHP-RNase (squares), BS-RNase (circles), monomeric LCCK-HP-RNase (triangles), or wild-type HP-RNase (rhombs). Cell survival values (means of triplicates) are expressed as percent of the corresponding values obtained in control cultures grown in the absence of protein. (A) Cell survival in the presence or in the absence of 50 μg/ml RNase. (B) Dose–response curves after 48 h of incubation.

Figure 4

Effects of dimeric HHP-RNase on normal HDFs (open squares), human papillary thyroid carcinoma cells TPC-1 (closed squares), and NPA (closed rhombs). Cell survival values, expressed as in Fig. 3, were calculated after 72 h of treatment.

Figure 5

Tumor cells sensitized to apoptosis by HHP-RNase. HDFs and human thyroid tumor (NPA) cells were grown, were exposed for 72 h to HHP-RNase (25 μg/ml), and were fixed and stained with Hoechst immunofluorescent reagent. (A) Untreated HDF cells. (B) HDF cells exposed to HHP-RNase. (C) Untreated NPA cells. (D) NPA cells exposed to HHP-RNase. The inset shows a fragmented and condensed apoptotic nucleus from an NPA treated cell. (A–D, ×360; D Inset, ×2,400.)