Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes.

HPV-16 E6 and E7 genes are required to efficiently immortalize a broad spectrum of cell types including cervical keratinocytes. Therefore, the E6/E7 genes can be considered relevant targets for anti-cancer therapy. We produced several engineered hairpin (HP) ribozymes to specifically disrupt HPV-16 E6/E7 mRNA. After extensive biochemical characterization, one anti-E6 HP ribozyme (R434) was selected for in vivo testing because of its superior catalytic capabilities. When expressed in cis, R434 efficiently inhibited E6 in vitro translation. Cis-expression of the HP ribozyme with HPV-16 E6/E7 genes in normal human keratinocytes reduced the growth rate and prevented immortalization. RNA analysis by reverse transcription-PCR showed that E6/E7 transcripts were cleaved in post-transfected cells and virtually were eliminated after long term expression. Of interest, an inactive version of the HP also was able to significantly affect the immortalizing ability of E6/E7, probably through passive hybridization. The combination of passive and cleaving antisense RNA therefore is established as an effective inhibitor of HPV-16 E6/E7 immortalization.

Kinetic characterization of human immunodeficiency virus type-1 protease-resistant variants.

Passage of human immunodeficiency virus type-1 (HIV-1) in T-lymphocyte cell lines in the presence of increasing concentrations of the hydroxylethylamino sulfonamide inhibitor VX-478 or VB-11328 results in sequential accumulation of mutations in HIV-1 protease. We have characterized recombinant HIV-1 proteases that contain these mutations either individually (L10F, M46I, I47V, I50V) or in combination (the double mutant L10F/I50V and the triple mutant M46I/I47V/I50V). The catalytic properties and affinities for sulfonamide inhibitors and other classes of inhibitors were determined. For the I50V mutant, the efficiency (kcat/Km) of processing peptides designed to mimic cleavage junctions in the HIV-1 gag-pol polypeptide was decreased up to 25-fold. The triple mutant had a 2-fold higher processing efficiency than the I50V single mutant for peptide substrates with Phe/Pro and Tyr/Pro cleavage sites, suggesting that the M46I and I47V mutations are compensatory. The effects of mutation on processing efficiency were used in conjunction with the inhibition constant (Ki) to evaluate the advantage of the mutation for viral replication in the presence of drug. These analyses support the virological observation that the addition of M46I and I47V mutations on the I50V mutant background enables increased survival of the HIV-1 virus as it replicates in the presence of VX-478. Crystal structures and molecular models of the active site of the HIV-1 protease mutants suggest that changes in the active site can selectively affect the binding energy of inhibitors with little corresponding change in substrate binding.

In vitro selection and characterization of human immunodeficiency virus type 1 (HIV-1) isolates with reduced sensitivity to hydroxyethylamino sulfonamide inhibitors of HIV-1 aspartyl protease.

Human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to the hydroxyethylamino sulfonamide protease inhibitors VB-11,328 and VX-478 have been selected in vitro by two independent serial passage protocols with HIV-1 in CEM-SS and MT-4 cell lines. Virus populations with greater than 100-fold-increased resistance to both inhibitors compared with the parental virus have been obtained. DNA sequence analyses of the protease genes from VB-11,328- and VX-478-resistant variants reveal a sequential accumulation of point mutations, with similar resistance patterns occurring for the two inhibitors. The deduced amino acid substitutions in the resistant protease are Leu-10-->Phe, Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val. This is the first observation in HIV protease resistance studies of an Ile-50-->Val mutation, a mutation that appears to arise uniquely against the sulfonamide inhibitor class. When the substitutions observed were introduced as single mutations into an HIV-1 infectious clone (HXB2), only the Ile-50-->Val mutant showed reduced sensitivity (two- to threefold) to VB-11,328 and VX-478. A triple protease mutant infectious clone carrying the mutations Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val, however, showed much greater reduction in sensitivity (14- to 20-fold) to VB-11,328 and VX-478. The same mutations were studied in recombinant HIV protease. The mutant protease Ile-50-->Val displays a much lower affinity for the inhibitors than the parent enzyme (< or = 80-fold). The protease triply mutated at Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val shows an even greater decrease in inhibitor binding (< or = 270-fold). The sulfonamide-resistant HIV protease variants remain sensitive to inhibitors from other chemical classes (Ro 31-8959 and L-735,524), suggesting possibilities for clinical use of HIV protease inhibitors in combination or serially.

Increased incidence of breast carcinoma in patients with irradiation for post-partum mastitis: a screening situation.

In Rochester, New York, 606 women were treated with ionizing radiation for post-partum mastitis, mostly between 1940 and 1955. Two-thirds of all breasts were treated, the average dose per breast being 377 rads (at 2.5 cm breast depth). Mammographic examinations were performed on 265 of these women still residing in this vicinity. Two nonpalpable carcinomas (with no axillary node metastases) were found in the twelve breast lesions that have been biopsied. Some of the biopsies revealed premalignant changes. It is recommended that women in this high-risk category have close medical supervision, as well as periodic mammographic evaluation, and that the importance of periodic breast self-examinations should be emphasized.