Isolation of efficient antivirals: genetic suppressor elements against HIV-1.

The development of general approaches for the isolation of efficient antivirals and the identification and validation of targets for drug screening are becoming increasingly important, due to the emergence of previously unrecognized viral diseases. The genetic suppressor element (GSE) technology is an approach based on the functional expression selection of efficient genetic inhibitors from random fragment libraries derived from a gene or genome of interest. We have applied this technology to isolate potent genetic inhibitors against HIV-1. Two strategies were used to select for GSEs that interfere with latent virus induction and productive HIV-1 infection based on the expression of intracellular and surface antigens. The selected GSEs clustered in seven narrowly defined regions of the HIV-1 genome and were found to be functionally active. These elements are potential candidates for the gene therapy of AIDS. The developed approaches can be applied to other viral pathogens, as well as for the identification of cellular genes supporting the HIV-1 life cycle.

Genetic suppressor elements: new tools for molecular oncology--thirteenth Cornelius P. Rhoads Memorial Award Lecture.

Genetic suppressor elements (GSEs) are short biologically active gene fragments that encode dominantly acting peptides or inhibitory antisense RNAs. GSEs can be isolated from a single gene or from a multigene complex by constructing a library of short random fragments of the target gene(s) in an expression vector, followed by expression selection for the desired phenotype in a suitable cellular system. GSE selection from a single gene allows one to develop efficient and specific inhibitors of the gene function and to identify functional protein domains. GSE selection from a multigene complex, such as a normalized (uniform abundance) cDNA population from mammalian cells, makes it possible to identify genes that are involved in selectable cellular phenotypes. The potential of GSE selection for uncovering novel gene functions was first demonstrated using bacteriophage lambda as a model system. GSE selection in retroviral expression vectors has been applied in mammalian cells to identify genes responsible for sensitivity to etoposide and other chemotherapeutic drugs. GSE selection is also useful for cloning and analysis of tumor suppressor genes and can be applied to identifying tumor-specific targets for future anticancer drugs. Investigators should find this experimental strategy applicable to many different areas of medical and biological research.

MDR-1 expression in metastatic malignant melanoma.

Metastatic malignant melanoma (MMM) carries an ominous prognosis. This poor prognosis is due in part to the remarkable resistance of MMM to a wide variety of antineoplastic agents. One common mechanism of resistance to chemotherapy is that of multidrug resistance (MDR) which is mediated by the MDR-1 gene. Previous efforts to determine MDR-1 expression in MMM have all failed to show expression using relatively insensitive immunohistochemical techniques. The purpose of this study is to evaluate the incidence of MDR-1 expression in MMM using a highly sensitive polymerase chain reaction (PCR)-based assay. Twenty-two clinical snap-frozen MMM specimens were analyzed. Total cellular RNA was extracted and quantitated from which cDNA was synthesized. Sequences of MDR-1 and beta 2-microglobulin (internal control) cDNA were coamplified using PCR. The relative yield of the MDR-1-specific PCR product was quantitated relative to that of a standard series of cell lines with known MDR-1 expression. Analysis revealed that 45% of MMM expressed MDR-1 at low levels. None of the tumors expressed high levels of MDR-1. Overall survival, disease-free survival, thickness of primary lesion, histology, and sex were not found to correlate with MDR-1 expression. We conclude that MDR-1 is frequently expressed at low levels in MMM. However, this incidence of MDR-1 expression suggests that non-MDR mechanisms of drug resistance are dominant in MMM. Further studies will be required to determine if MDR-1 expression is of clinical significance in MMM.

Clinical correlates of MDR1 (P-glycoprotein) gene expression in ovarian and small-cell lung carcinomas.

BACKGROUND: Expression of the MDR1 (P-glycoprotein) gene causes resistance to several classes of lipophilic anti-cancer drugs, but MDR1 expression in untreated ovarian and lung carcinomas is rarely detectable by standard assays. PURPOSE: This study was designed to measure the MDR1 messenger RNA (mRNA) content of ovarian and lung carcinomas and to analyze clinical correlations of MDR1 expression in these tumors. METHODS: A sensitive assay based on the polymerase chain reaction (PCR) was used in a retrospective study to measure MDR1 mRNA in biopsy samples of 100 solid tumors, including 60 ovarian and 32 lung carcinomas. The levels of MDR1 mRNA were correlated with history of chemotherapeutic treatment for all tumors; for ovarian and small-cell lung carcinomas (SCLCs), these levels were also correlated with subsequent tumor response to chemotherapy. RESULTS: Among previously untreated patients, MDR1 mRNA was expressed in 68% (50 of 74) of all tumors. Among patients pretreated with chemotherapy regimens that included at least one P-glycoprotein-transported drug (MDR regimens), 95% (20 of 21) of all tumors expressed MDR1 mRNA though the incidence of high-level MDR1 expression was decreased among the treated tumors. MDR1 mRNA was expressed in only one of five tumors treated with regimens that included no P-glycoprotein substrates (non-MDR regimens). Subsequent tumor response to chemotherapy was evaluated in 35 patients with ovarian carcinoma and seven patients with SCLC. The presence of even very low levels of MDR1 mRNA correlated with the lack of response to MDR regimens in these tumor types (P < .035 for ovarian carcinomas, P < .029 for SCLCs, and P < .0005 for both tumor types; Fisher's Exact Test). CONCLUSIONS: Low-level expression of MDR1 mRNA correlates with clinical resistance to combination chemotherapy in ovarian cancer and SCLC. We hypothesize that MDR1 is expressed in a subpopulation of more malignant tumor cells possessing multiple mechanisms of drug resistance. IMPLICATIONS: The presence of MDR1-expressing tumor cells may be useful as a predictive marker for clinical resistance to combination chemotherapy in ovarian cancer and SCLC. Prospective studies are needed to confirm this hypothesis.

Isolation of dominant negative mutants and inhibitory antisense RNA sequences by expression selection of random DNA fragments.

Selective inhibition of specific genes can be accomplished using genetic suppressor elements (GSEs) that encode antisense RNA, dominant negative mutant proteins, or other regulatory products. GSEs may correspond to partial sequences of target genes, usually identified by trial and error. We have used bacteriophage lambda as a model system to test a concept that biologically active GSEs may be generated by random DNA fragmentation and identified by expression selection. Fragments from eleven different regions of lambda genome, encoding specific peptides or antisense RNA sequences, rendered E. coli resistant to the phage. Analysis of these GSEs revealed some previously unknown functions of phage lambda, including suppression of the cellular lambda receptor by an 'accessory' gene of the phage. The random fragment selection strategy provides a general approach to the generation of efficient GSEs and elucidation of novel gene functions.

Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction.

The resistance of tumor cells to chemotherapeutic drugs is a major obstacle to successful cancer chemotherapy. In human cells, expression of the MDR1 gene, encoding a transmembrane efflux pump (P-glycoprotein), leads to decreased intracellular accumulation and resistance to a variety of lipophilic drugs (multidrug resistance; MDR). The levels of MDR in cell lines selected in vitro have been shown to correlate with the steady-state levels of MDR1 mRNA and P-glycoprotein. In cells with a severalfold increase in cellular drug resistance, MDR1 expression levels are close to the limits of detection by conventional assays. MDR1 expression has been frequently observed in human tumors after chemotherapy and in some but not all types of clinically refractory tumors untreated with chemotherapeutic drugs. We have devised a highly sensitive, specific, and quantitative protocol for measuring the levels of MDR1 mRNA in clinical samples, based on the polymerase chain reaction. We have used this assay to measure MDR1 gene expression in MDR cell lines and greater than 300 normal tissues, tumor-derived cell lines, and clinical specimens of untreated tumors of the types in which MDR1 expression was rarely observed by standard assays. Low levels of MDR1 expression were found by polymerase chain reaction in most solid tumors and leukemias tested. The frequency of samples without detectable MDR1 expression varied among different types of tumors; MDR1-negative samples were most common among tumor types known to be relatively responsive to chemotherapy.

Bacteriophages of Bordetella sp.: features of lysogeny and conversion.

It has been the purpose of this paper to study molecular-biological features of the Bordetella bacteriophage interaction with the host cell during lysogeny and conversion as well as to determine the degree of homology between genomes of homologous and heterologous bacteriophages. Genomes of bacteriophages from B. pertussis 134, 41405 and B. bronchiseptica 214 were studied. Heteroduplex and restriction analyses revealed a heterogeneity of bacteriophage populations, and their DNAs were found to differ in size and position of inserts. As shown by blot hybridization, the bacteriophage genome is not inserted into the chromosome of the lysogenic cell but apparently exists as an autonomous plasmid replicon. It has been established that during conversion only a part of the phage genome is inserted into the chromosome of the recipient cell.

All species of the genus Bordetella contain genes for pertussis toxin of Bordetella pertussis.

A molecular probe for the PT-operon of B. pertussis hybridized with 4.7 Kb EcoRI-fragments of chromosomal DNAs of B. pertussis strain 475 phase I, phase IV, B. parapertussis strains 504 and 17903, B. bronchiseptica strain 214, B. parapertussis strain 17903-convertant of B. pertussis phage 134 but not with phage 134 DNA under stringent conditions of DNA-DNA hybridization. This fact indicates the presence of PT-genes in all Bordetella species. Since there is no production of PT in B. parapertussis and B. bronchiseptica, a presence of regulatory mutations in the PT-operon or absence of the functionally active vir-gene product in these species is suggested.