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Context: Loss of viral suppression in patients infected with human immunodeficiency virus (HIV), who are receiving potent antiretroviral therapy, has been attributed to outgrowth of drug-resistant virus; however, resistance patterns are not well characterized in patients whose protease inhibitor combination therapy fails afterachieving viral suppression.Objective: To characterize drug susceptibility of virus from HIV-infected patients who are failing to sustain suppression while taking an indinavir-containing antiretroviral regimen.Design and Setting: Substudy of the AIDS Clinical Trials Group 343, a multicenter clinical research trial conducted between February 1997 and October 1998.Patients: Twenty-six subjects who experienced rebound (HIV RNA level > or =200 copies/mL) during indinavir monotherapy (n = 9) or triple-drug therapy (indinavir, lamivudine, and zidovudine; n = 17) after initially achieving suppression while receiving all 3 drugs, and 10 control subjects who had viral suppression while receiving triple-drug therapy.Main Outcome Measure: Drug susceptibility, determined by a phenotypic assay and genotypic evidence of resistance assessed by nucleotide sequencing of protease and reverse transcriptase, compared among the 3 patient groups.Results: Indinavir resistance was not detected in the 9 subjects with viral rebound during indinavir monotherapy or in the 17 subjects with rebound during triple-drug therapy, despite plasma HIV RNA levels ranging from 10(2) to 10(5) copies/mL. In contrast, lamivudine resistance was detected by phenotypic assay in rebound isolates from 14 of 17 subjects receiving triple-drug therapy, and genotypic analyses showed changes at codon 184 of reverse transcriptase in these 14 isolates. Mean random plasma indinavir concentrations in the 2 groups with rebound were similar to those of a control group with sustained viral suppression, although levels below 50 ng/mL were more frequent in the triple-drug group than in the control group (P = .03).Conclusions: Loss of viral suppression may be due to suboptimal antiviral potency, and selection of a predominantly indinavir-resistant virus population may be delayed for months even in the presence of ongoing indinavir therapy. The results suggest possible value in assessing strategies using drug components of failing regimens evaluated with resistance testing. [ABSTRACT FROM AUTHOR]

Cross-resistance between zidovudine, stavudine, and lamivudine was studied, using purified recombinant reverse transcriptase from a zidovudine-susceptible and -resistant pair of clinical isolates of human immunodeficiency virus type 1. The zidovudine-resistant isolate exhibited low-level cross-resistance to both stavudine and lamivudine in drug susceptibility assays. Enzyme from the resistant isolate demonstrated reduced inhibition by zidovudine triphosphate and stavudine triphosphate and, to a lesser extent, lamivudine triphosphate. These findings provide additional evidence at the viral and enzyme level for cross-resistance between zidovudine and stavudine, and they suggest a possible effect of zidovudine resistance on susceptibility to lamivudine.

Objective(s): To compare antiretroviral resistance susceptibility testing of patient HIV-1 strains using genotype and phenotype methods., Design: Eighteen plasma samples with viral load > 2000 HIV-1 RNA copies/ml were randomly selected for testing by both methods. Disease and treatment data were available for all patients., Methods: Samples were analysed genotypically using a kit assay (HIV-1 Genotyping Systems, Applied Biosystems), performed by the Clinical Research Laboratory at Macfarlane Burnet Centre for Medical Research. Samples were analysed phenotypically using a rapid phenotypic assay (PhenoSenseTM HIV, ViroLogic), performed by the manufacturer. Results from both methods were interpreted using a defined protocol. Each susceptibility assay was performed and interpreted by individuals unaware of either the clinical data or the results of the other susceptibility assay. Concordance was defined categorically as either the presence of reduced susceptibility (> 2.5-fold change) in the phenotypic assay and resistance associated mutations in the genotypic assay, or the absence of these findings in both assays., Results: Concordance between phenotypic and genotypic susceptibility testing was 81% for nucleoside reverse transcriptase inhibitors, 91% for non-nucleoside reverse transcriptase inhibitors and 90% for protease inhibitors. Complete concordance between phenotype and genotype for all 14 drugs evaluated was observed in three (17%) patient samples., Conclusions: Phenotypic and genotypic susceptibility appear to provide similar results. However, interpretation of genotypic results can be complicated, and both methods still require clinical validation.

Objective: Structured antiretroviral treatment interruption (STI) has been advocated as a therapeutic strategy for HIV-1 infection. We report initial observations of cerebrospinal fluid (CSF) HIV-1 infection in five patients undergoing serial lumbar punctures (LPs) during STI undertaken following virological failure., Design and Methods: In this prospective observational study we quantified HIV-1 RNA concentrations and assessed both phenotypic drug susceptibility profiles and genotypic antiviral drug resistance mutations in CSF and plasma during the period of treatment interruption. CSF white blood cells were also counted, and patients' neurological status monitored., Results: In four of the patients, CSF HIV-1 concentration increased more rapidly than that of the plasma, with consequent reduction in the ratio between plasma and CSF viral loads (pVL : cVL). Three individuals developed robust, though asymptomatic CSF lymphocytic pleocytosis. In all patients the predominant HIV-1 quasispecies shifted simultaneously in CSF and plasma from a drug-resistant to a more drug-susceptible phenotype with identical and simultaneous changes in genotypes associated with drug resistance., Conclusions: STI may be accompanied by previously unrecognized changes in tissue viral exposures and lymphocyte traffic. Hence, despite 'virological failure' as evidenced by persistent plasma viremia, ongoing antiretroviral treatment prior to its interruption appeared to suppress CSF HIV-1 infection (indeed more effectively than that of plasma) and restrain lymphocyte traffic into the CSF. Simultaneous change of resistance mutations in CSF and plasma was likely due to re-emergence and overgrowth of pre-existing strains with ready exchange of virus between these two compartments, either facilitated by or provoking a local CSF lymphocytosis.

To confirm the vertical transmission of multidrug-resistant (MDR) human immunodeficiency virus type 1 (HIV-1) and to assess its impact on further evolution of drug-resistant virus in an infant, proviral DNA amplified from infected peripheral blood mononuclear cell cultures was sequenced to identify reverse transcriptase (RT) and protease (PR) mutations. The infant had proviral DNA with evidence of RT mutations (M41L, L74V, and T215Y) and 3 PR substitutions (K20R, M36I, and V82A). After delivery, the mother's proviral DNA had the same substitutions. Phylogenetic analyses of these HIV-1 RT and PR sequences indicated epidemiological linkage. Plasma drug susceptibility was determined by using a recombinant virus assay. Plasma HIV-1 obtained after the infant's birth demonstrated reduced susceptibility to zidovudine and ritonavir. Thus, vertical transmission of MDR HIV-1 was demonstrated in the setting of detectable maternal plasma viremia. Further accumulation of broad MDR in the infant's virus to 3 antiretroviral classes occurred, despite postnatal therapy.

Background: In many patients with human immunodeficiency virus (HIV) infection, therapy with potent antiretroviral drugs does not result in complete suppression of HIV replication. The effect of cessation of therapy in these patients is unknown., Methods: Sixteen patients who had a plasma HIV RNA level of more than 2500 copies per milliliter during combination antiretroviral-drug therapy were randomly assigned, in a 2:1 ratio, to discontinue or continue therapy. Plasma HIV RNA levels, CD4 cell counts, and drug susceptibility were measured weekly. Viral replicative capacity was measured at base line and at week 12., Results: Discontinuation of therapy for 12 weeks was associated with a median decrease in the CD4 cell count of 128 cells per cubic millimeter and an increase in the plasma HIV RNA level of 0.84 log copies per milliliter. Virus from all patients with detectable resistance at entry became susceptible to HIV-protease inhibitors within 16 weeks after the discontinuation of therapy. Drug susceptibility began to increase a median of six weeks after the discontinuation of therapy and was temporally associated with increases in plasma HIV RNA levels and decreases in CD4 cell counts. Viral replicative capacity, measured by means of a recombinant-virus assay, was low at entry into the study and increased after therapy was discontinued. Despite the loss of detectable resistance in plasma, resistant virus was cultured from peripheral-blood mononuclear cells in five of nine patients who could be evaluated. Plasma HIV RNA levels, CD4 cell counts, and drug susceptibility remained stable in the patients who continued therapy., Conclusions: Despite the presence of reduced drug susceptibility, antiretroviral-drug therapy can provide immunologic and virologic benefit. This benefit reflects continued antiviral-drug activity and the maintenance of a viral population with a reduced replicative capacity.

Background: Clinical studies have demonstrated a correlation between the response to second-line antiretroviral therapy and the number of drugs in the regimen to which the virus is susceptible. These studies have largely been performed in patients with viral loads over 1000 copies/ml., Objectives: To examine the evolution of resistance during early virological failure, and the potential role of susceptibility testing in patients with low viral loads (below 1000 copies/ml), in treatment-experienced patients., Methods: Drug susceptibility and genotypes of HIV-1 from indinavir-experienced patients undergoing therapy with nelfinavir, saquinavir, abacavir and either a second nucleoside reverse transcriptase inhibitor (NRTI) or nevirapine were determined., Results: Sixteen subjects were studied. Five of the ten subjects treated with nevirapine, and one of six treated with a second NRTI, achieved and maintained plasma HIV RNA < 500 copies/ml. Virus from the treatment failures lost susceptibility to one or more treatment drugs, including nelfinavir and/or saquinavir, after 4 to 36 weeks of treatment. In six of the ten failures, virus with new reductions in drug susceptibility was detected prior to failure. In five of the six failures who had at least one plasma sample with a viral load between 50 and 1000 copies/ml, reductions in susceptibility to one or more treatment drugs were detected (viral load range: 260 to 630 copies/ml)., Conclusions: Drug resistance can be detected at viral loads below 1000 copies/ml which may be predictive of treatment failure. Failure of a second line regimen was typically associated with early evolution of resistance in HIV protease.

Recently, significant numbers of individuals with primary human immunodeficiency virus (HIV) infection have been found to harbor viral strains with reduced susceptibility to antiretroviral drugs. In one study, HIV from 16% of such antiretroviral-naive individuals was shown to have a susceptibility to nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) between 2.5- and 10-fold lower than that of a wild-type control. Mutations in the RT domain that had previously been associated with antiretroviral resistance were not shared by these strains. We have analyzed by logistic regression 46 variable amino acid sites in RT for their effect on susceptibility and have identified two novel sites influencing susceptibility to NNRTIs: amino acids 135 and 283 in RT. Eight different combinations of amino acids at these sites were observed among these patients. These combinations showed a 14-fold range in mean susceptibility to both nevirapine and delavirdine. In vitro mutagenesis of the control strain combined with a phenotypic assay confirmed the significance of amino acid variation at these sites for susceptibility to NNRTIs.

The extent to which human immunodeficiency virus (HIV) type 1 drug resistance compromises therapeutic efficacy is intimately tied to drug potency and exposure. Most HIV-1 protease inhibitors maintain in vivo trough levels above their human serum protein binding-corrected IC(95) values for wild-type HIV-1. However, these troughs are well below corrected IC(95) values for protease inhibitor-resistant viruses from patients experiencing virologic failure of indinavir and/or nelfinavir. This suggests that none of the single protease inhibitors would be effective after many cases of protease inhibitor failure. However, saquinavir, amprenavir, and indinavir blood levels are increased substantially when each is coadministered with ritonavir, with 12-h troughs exceeding corrected wild-type IC(95) by 2-, 7-, and 28-79-fold, respectively. These indinavir and amprenavir troughs exceed IC(95) for most protease inhibitor-resistant viruses tested. This suggests that twice-daily indinavir-ritonavir and, to a lesser extent, amprenavir-ritonavir may be effective for many patients with viruses resistant to protease inhibitors.

To assess the prevalence of mutations associated with decreased antiretroviral drug susceptibility, specimens were tested from persons infected with human immunodeficiency virus (HIV) during 1993-1998. Subjects were drug naive and were attending sexually transmitted disease clinics in 6 US cities. All were enrolled consecutively and had tested negative for HIV during the 2 years before enrollment. Plasma specimens from patients having >/=1 reverse transcriptase (RT) or primary protease mutation were tested phenotypically with a recombinant virus assay. Of 99 patients, 6 (6%) had mutations associated with zidovudine resistance, 2 (2%) had mutations associated with nonnucleoside RT inhibitor resistance, and 1 (1%) had a primary protease mutation. Overall, the prevalence of resistance-associated primary mutations was 5%, although high levels of decreased drug susceptibility (IC(50)s >/=10 times that of a reference virus) were observed in just 1%. These findings confirm the transmission of these mutations to drug-naive persons.

Amprenavir (Agenerase, 141-W94, VX-478) is a human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PRI) recently approved for the treatment of HIV-1 infection in the United States. A major cause of treatment failure is the development of resistance to PRIs. One potential use for amprenavir is as salvage therapy for patients for whom treatment that includes one (or more) of the other four currently approved PRIs-saquinavir, indinavir, ritonavir, and nelfinavir-has failed. We evaluated the cross-resistance to amprenavir of viruses that evolved during treatment with the two most commonly prescribed PRIs, nelfinavir and indinavir. Unexpectedly, a dramatic increase in susceptibility (2.5- to 12. 5-fold) was observed with 20 of 312 (6.4%) patient viruses analyzed. The most pronounced increases in susceptibility were strongly associated with an N88S mutation in protease. All viruses that carried the N88S mutation were hypersensitive to amprenavir. Site-directed mutagenesis studies confirmed the causal role of N88S in determining amprenavir hypersensitivity. The presence of the N88S mutation and associated amprenavir hypersensitivity may be useful in predicting an improved clinical response to amprenavir salvage therapy.

Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. We have developed a novel phenotypic drug susceptibility assay that may be useful in guiding therapy and improving long-term suppression of HIV replication. Susceptibility to protease (PR) and reverse transcriptase (RT) inhibitors is measured by using resistance test vectors (RTVs) that contain a luciferase indicator gene and PR and RT sequences derived from HIV-1 in patient plasma. Cells are transfected with RTV DNA, resulting in the production of virus particles that are used to infect target cells. Since RTVs are replication defective, luciferase activity is measured following a single round of replication. The assay has been automated to increase throughput and is completed in 8 to 10 days. Test results may be useful in facilitating the selection of optimal treatment regimens for patients who have failed prior therapy or drug-naive patients infected with drug-resistant virus. In addition, the assay can be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant strains of HIV-1.

Chinese hamster ovary cells used for pharmaceutical protein production express non-infectious retrovirus-like particles. To assure the safety of pharmaceutical proteins, validation of the ability of manufacturing process to clear retrovirus-like particles is required for product registration. Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for validation studies. Some chromatography procedures used for pharmaceutical protein purification utilize low pH (< pH 4.0) elution buffers which readily inactivate X-MuLV. Therefore, cell-based infectivity assays are unable to evaluate the physical removal of X-MuLV by these chromatography procedures. To distinguish viral inactivation by low pH treatment from viral removal by chromatography, a quantitative competitive reverse transcription PCR method capable of quantifying both infectious and non-infectious X-MuLV has been developed. This method quantifies X-MuLV particles in chromatography pools by quantifying the X-MuLV particle RNA (pRNA). The difference between the amount of X-MuLV pRNA in the load pool and the product-containing elution pool represents the extent of X-MuLV removal. This method is an extremely powerful complement to cell based-infectivity assays as it allows physical removal of X-MuLV by chromatography and filtration procedures to be distinguished from X-MuLV inactivation when buffers with the ability to inactivate retrovirus are used.

Context: The transmission of drug-resistant human immunodeficiency virus (HIV) has been documented, but the prevalence of such transmission is unknown., Objective: To assess the spectrum and frequency of antiretroviral susceptibility among subjects with primary HIV infection., Design, Setting, and Patients: Retrospective analysis of 141 subjects identified from clinical research centers in 5 major metropolitan areas, enrolled from 1989 to 1998, with HIV seroconversion within the preceding 12 months and no more than 7 days' prior antiretroviral (ARV) therapy., Main Outcome Measures: Phenotypic and genotypic ARV susceptibility of HIV from plasma samples., Results: The transmission of drug-resistant HIV as assessed by a greater than 10-fold reduction in ARV susceptibility to 1 or more drugs was observed in 3 (2%) of 141 subjects, including to a nonnucleoside reverse transcriptase inhibitor in 1 patient and to a nucleoside reverse transcriptase inhibitor and a protease inhibitor in 2 patients. Population-based sequence analysis of these 3 samples identified multidrug-resistance mutations in reverse transcriptase (M184V, T215Y, K219K/R) and protease (L101/V, K20R, M361, M46I, G48V, L63P, A71T, V771, V82T, 184V, L90M) in the 2 latter patient samples, along with numerous polymorphisms. A reduction in susceptibility of greater than 2.5- to 10-fold to 1 or more drugs was observed in viral isolates from 36 patients (26%). Sequence analysis of these 36 samples identified well-characterized drug resistance mutation in reverse transcriptase and protease in only 1 of these patients., Conclusions: Reductions in drug susceptibility of more than 10-fold were rare among this cohort of recently HIV-infected subjects and were distributed among each of the 3 major classes of ARV drugs tested. Reductions in susceptibility of more than 2.5- to 10-fold to certain ARV drugs of unknown clinical significance were highly prevalent among newly infected patients. Resistance testing may be warranted to monitor the frequency of drug resistance over time and to assess the potential for geographic variability.

The emergence of drug-resistant human immunodeficiency virus type 1 is a frequent cause of failure of combination therapies comprising reverse transcriptase and protease inhibitors. Rational design of salvage therapies requires new methods to assess drug susceptibility. A novel phenotypic drug susceptibility assay was developed and used to measure the drug susceptibilities of viruses obtained from 2 patients treated with zidovudine, lamivudine, and nelfinavir. Results showed that phenotypic drug resistance may be detectable before virus load rebound, treatment failure does not always imply viral resistance to all drugs in a treatment regimen, and persons with similar antiviral treatment histories and clinical courses may have different phenotypic drug resistance profiles at the time that treatment fails.

Twenty human immunodeficiency virus-infected patients experiencing virologic failure of an indinavir- or ritonavir-containing treatment regimen were evaluated in a prospective, open-label study. Subjects received nelfinavir, saquinavir, abacavir, and either another nucleoside analog (n=10) or nevirapine (n=10). Patients treated with the nevirapine-containing regimen experienced significantly greater virologic suppression at week 24 than those not treated with nevirapine (P=.04). Baseline phenotypic drug susceptibility was strongly correlated with outcome in both treatment arms. Subjects with baseline virus phenotypically sensitive to 2 or 3 drugs in the salvage regimen experienced significantly greater virus load suppression than those with baseline virus sensitive to 0 or 1 drug (median week-24 change=-2.24 log and -0.35 log, respectively; P=.01). In conclusion, non-nucleoside reverse transcriptase inhibitors may represent a potent drug in salvage therapy regimens after failure of an indinavir or ritonavir regimen. Phenotypic resistance testing may provide a useful tool for selecting more effective salvage regimens.

The Chinese hamster cell lines E36 and CHOK1 dramatically differ in susceptibility to amphotropic murine leukemia virus (A-MuLV) and gibbon ape leukemia virus (GALV); E36 cells are highly susceptible to both viruses, CHOK1 cells are not. We have previously shown that GALV can infect E36 cells by using both its own receptor, HaPit1, and the A-MuLV receptor, HaPit2. Given that the two cell lines are from the same species, the loss of function of both of these receptors in CHOK1 cells is surprising. Other studies have shown that CHOK1 cells secrete proteins that block A-MuLV entry into CHOK1 as well as E36, suggesting the two A-MuLV receptors are functionally identical. However, CHOK1 conditioned medium does not block GALV entry into E36, indicating the secreted inhibitors do not block HaPit1. HaPit1 and ChoPit1 therefore differ as receptors for GALV; ChoPit1 is either inactivated by secreted factors or intrinsically nonfunctional. To determine why GALV cannot infect CHOK1, we cloned and sequenced ChoPit1 and ChoPit2. ChoPit2 is almost identical to HaPit2, which explains why CHOK1 conditioned medium blocks A-MuLV entry via both receptors. Although ChoPit1 and HaPit1 are 91% identical, a notable difference is at position 550 in the fourth extracellular region, shown by several studies to be crucial for GALV infection. Pit1 and HaPit1 have aspartate at 550, whereas ChoPit1 has threonine at this position. We assessed the significance of this difference for GALV infection by replacing the aspartate 550 in Pit1 with threonine. This single substitution rendered Pit1 nonfunctional for GALV and suggests that threonine at 550 inactivates ChoPit1 as a GALV receptor. Whether native ChoPit1 functions for GALV was determined by interference assays using Lec8, a glycosylation-deficient derivative of CHOK1 that is susceptible to both viruses and that has the same receptors as CHOK1. Unlike with E36, GALV and A-MuLV exhibited reciprocal interference when infecting Lec8, suggesting that they use the same receptor. We conclude both viruses can use ChoPit2 in the absence of the inhibitors secreted by CHOK1 and ChoPit1 is nonfunctional.

The development of 5' nuclease assays represents a significant advance in nucleic acid quantitation. This approach utilizes the 5'-3' exonuclease activity of Thermus aquaticus (Taq) polymerase to cleave a dual-labelled probe annealed to a target sequence during amplification. The release of a fluorogenic tag from the 5' end of the probe is proportional to the target sequence concentration (copy number), and can be measured either at endpoint (post-amplification), or in real time', where the increase in emission intensity is followed on a per-cycle basis.

Linear peptides derived from the membrane proximal region of the gp41 ectodomain are effective inhibitors of HIV type 1 (HIV-1)-mediated fusion events. These inhibitory peptides lack structure in solution, rendering mechanistic interpretation of their activity difficult. Using structurally constrained analogs of these molecules, we demonstrate that the peptides inhibit infectivity by adopting a helical conformation. Moreover, we show that a specific face of the helix must be exposed to block viral infectivity. Recent crystal structures show that the region of gp41 corresponding to the inhibitory peptides is helical and uses the analogous face to pack against a groove formed by an N-terminal coiled-coil trimer. Our results provide a direct link between the inhibition of HIV-1 infectivity by these peptides and the x-ray structures, and suggest that the conformation of gp41 observed by crystallography represents the fusogenic state. Other agents that block HIV-1 infectivity by binding to this groove may hold promise for the treatment of AIDS.

To gain a more complete understanding of c-myc regulation in chickens, we have completed the structural characterization of the chicken c-myc gene and have begun to investigate c-myc transcription and protein expression. A comparison of c-myc structure and expression between mammals and birds presents an enigma: there are striking similarities in the pattern of gene expression in the absence of obvious sequence similarities in the controlling elements. We have begun to investigate c-myc and v-myc function using retroviral vectors that differ solely in the Myc proteins that they express. We show that while the overexpression of the smaller c-Myc protein is sufficient to induce morphological transformation in chicken embryo fibroblasts, overexpression of v-Myc provides a stronger signal for cells to enter the cell cycle and is a more potent inducer of apoptosis than c-Myc.

This paper addresses seven questions with respect to the transfer, integration, amplification and genetic stability of recombinant DNA in mammalian genomes. Most of these questions were raised with those issues in mind which are frequently discussed in the context of the manufacture of biologicals of therapeutic value on the basis of recombinant cell lines. For the most part, a high degree of ignorance has to be acknowledged and only very limited fragments of information are available. The reason for this ignorance is, as will become clear from this article, the sheer size and complexity of the mammalian genome and the inadequacy of presently available tools to unravel this complexity.

To analyze the relationship between c-myc gene expression and chondrocyte proliferation and maturation during endochondral ossification, Day 18-19 chick embryo sterna were pulse-labeled with [3H]thymidine, and serial sections were processed for autoradiography and in situ hybridization. Proliferating chondrocytes, located in four distinct areas of the developing sternum, all contained high levels of c-myc transcripts, whereas postmitotic chondrocytes (such as hypertrophic chondrocytes) contained undetectable amounts. These findings were confirmed by Northern blot analysis and by the observation that antisense c-myc oligomer treatment inhibited proliferation in cultured chondrocytes. Constitutive overexpression of c-myc by retroviral vectors in immature chondrocyte cultures (c-myc cultures) maintained the cells in a proliferative state and blocked their maturation into hypertrophic chondrocytes. The lack of maturation in the c-myc cultures was corroborated by analysis of type X collagen gene regulation. Control immature cultures contained strong repressor activity for the type X collagen gene promoter, as revealed by transfection assays; repressor activity was lost upon maturation and activation of type X collagen synthesis. In the c-myc cultures, however, repressor activity persisted. Thus, c-myc participates in the normal changes in proliferation accompanying chondrocyte maturation in vivo and in culture. The decreases in c-myc expression and cell proliferation appear to be required for completion of maturation.

Continued exposure of rats to carcinogenic doses of methapyrilene (MP) leads to elevated levels of 5-methyl-deoxycytidine (5MC) in liver DNA. Since gene expression often correlates with DNA methylation, we investigated these parameters in the MP-induced hepatocellular carcinomas of Fischer 344 rats. DNA was hypermethylated in liver tissue surrounding the tumors relative to liver tissue of untreated controls of the same age, while tumor DNA was not; DNA methylation declined to normal levels when MP treatment ceased. Gene expression analysis showed measurable levels of mRNA for c-Ki-ras, erb-B, erb-B2, hck, src, lyn, vav, trk, raf-1, l-myc, c-jun, c-yes, c-myc, c-abl, and p53. No significant differences in expression for these and other oncogenes were seen between tumors and surrounding livers, although erb-B2 and vav showed visible decreases compared with normal liver. Hypermethylation of DNA and expression of these oncogenes in MP-treated tissues were not correlated. Levels of mRNA for the same genes in MP-treated hepatocytes in culture were similar to in vivo levels; analysis of DNA synthesis levels showed that this gene expression pattern occurred in the absence of proliferation bursts or toxicity in these cells, thus suggesting that treatment in vivo may produce the same results.

Strain improvement of agriculturally important animals will require efficient techniques for gene delivery, the ability to regulate the expression of the newly introduced genes and, most important, the identification of genes whose appropriate expression could cause improvement of the animal. We have developed a series of avian retroviral vectors that can be used to introduce new genetic information into the germ line of chickens, for which transgenics cannot be created by direct microinjection of DNA into fertilized eggs. We have identified a 220-bp segment of the chicken skeletal muscle alpha-actin gene that can cause other genes to be expressed specifically in striated muscle. This chicken promoter shows correct tissue specificity in transgenic mice and presumably could be used in other mammalian species. The skeletal muscle alpha-actin promoter has been inserted into the avian retroviral vectors and the promoter is functional in cultured cells infected by these retroviral vectors. The tissue specificity of the expression of the skeletal muscle alpha-actin promoter carried by the retroviral vectors will soon be tested in vivo. We are studying two types of genes that might be useful in strain improvement; genes that could produce dominant resistance to infection by pathogenic viruses, and genes that could play critical roles in muscle development. Expression of the envelope glycoprotein of retroviruses can specifically block the cellular receptor that viruses use to infect a susceptible cell. Expression of the avian leukosis virus subgroup A envelope in transgenic chickens prevents infection by pathogenic viruses of the same subgroup. We are attempting to block reticuloendotheliosis virus infection by expressing the reticuloendotheliosis envelope glycoprotein. We have shown that we can block infection in cultured cells, and we are now creating retroviral vectors for experiments in vivo. We have also begun to study the cellular homologue of the ski oncogene, which has been shown to stimulate the differentiation of quail myoblasts in vitro. Biologically active cDNAs have been isolated; we have now begun to analyse the effects of expressing the c-ski proteins in the whole animal.

To investigate the degree of homology which may exist between rat liver RNA populations during development, regeneration, and neoplasia, we hybridized polyadenylated RNAs from (a) normal adult, (b) 24-h regenerating, (c) 20-day fetal livers, and (d) the transplantable Morris hepatoma 5123tc to homologous and heterologous complementary DNAs and to cDNAs enriched for sequences preferentially transcribed in either adult or fetal liver. We also compared the in vitro translation products of these RNAs. Analyses of normal adult, regenerating, and fetal liver RNA populations and their translation products show that the overall pattern of gene expression during liver regeneration differs little from that of normal adult rat liver and that mature hepatocytes do not appear to revert to an "immature" state upon reentering the cell cycle. Comparisons between fetal, normal adult, and tumor RNA populations revealed that RNA populations from fetal liver and the 5123tc tumor lack sequences normally expressed in the mature adult liver. However, the tumor does not "reexpress" sequences which are preferentially expressed in fetal livers.

We analyzed in isolated rat liver cell populations and in fetal and neoplastic livers the distribution of RNA sequences which hybridize with alpha-fetoprotein (AFP) complementary DNA clones. Parenchymal and nonparenchymal cell populations were isolated from normal, regenerating, preneoplastic, and bile duct-ligated rat livers. We found that oval cells, fetal liver, and a primary hepatocellular carcinoma contain the full length 2.3-kilobase AFP messenger RNA (mRNA); in normal adult rat liver, 2.3-kilobase AFP mRNA is found at low levels in an unidentified subpopulation of nonparenchymal cells but is not detected in hepatocytes; both parenchymal and nonparenchymal cells from normal or preneoplastic livers contain in variable proportion a smaller AFP RNA which hybridizes only with complementary DNA clones containing sequences located near the 5' end of the rat AFP gene; during liver regeneration induced by CCl4, elevation of the full length AFP mRNA occurs in nonparenchymal cells but seemingly not in hepatocytes. The results suggest that some cells in the nonparenchymal cell fraction of normal adult rat liver might retain the capacity to produce the 2.3-kilobase AFP mRNA found in large amounts in fetal livers, oval cells, and hepatic tumors. Although the nature of these cells remains to be determined, we suggest that such cells might be the source of the small amounts of AFP synthesized in normal rat liver and may constitute the proposed but as yet uncharacterized "facultative stem cell" compartment in rat liver.

We examined the homology between polysomal polyadenylated ribonucleic acid (mRNA) populations of hepatoma 252, a tumor which is deficient in the synthesis of plasma proteins, and those of normal and regenerating rat liver. Hybridization of polyadenylated mRNA populations with homologous or heterologous complementary deoxyribonucleic acids showed that mRNA from total and free polysomes from hepatoma 252 lack sequences which are present in normal or regenerating liver. Although there are obvious differences in the abundance of sequences between tumor and normal or regenerating liver polysomal mRNA, we did not detect, with the techniques used in this work, tumor-specific sequences. Analysis of hybridization curves using derivative plots did not reveal the presence in tumor mRNA of a high complexity class not present in normal liver. We conclude that alterations in mRNA populations of free and total polysomes of this tumor primarily reflect processes of genetic restriction rather than the derepression of previously unexpressed genes.

The number of transcripts of the cellular oncogene ras, which is homologous to the transforming gene of Harvey sarcoma virus, increases during liver regeneration in rats. The increase in these transcripts in liver polysomal polyadenylated RNA occurs at the time of activation of DNA synthesis during the regenerative process induced by partial hepatectomy or carbon tetrachloride injury. The number of ras transcripts returns to basal levels within 72 hours. These observations show that transcription of a cellular oncogene increases in a regulated way in a nonneoplastic growth process.

We examined the methylation pattern and organization of the AFP gene in whole livers and in isolated cell populations purified from livers of rats fed a carcinogenic diet which interferes with DNA methylation. Using restriction endonuclease digestion, we find no differences in methylation pattern and overall organization of the AFP gene in oval cells (AFP-producers) and hepatocytes (non-producers) isolated at the early stages of carcinogenesis. Our studies indicate that in cell populations which produce AFP as well as in cells which are not active in AFP synthesis, the majority of the CCGG sites of the AFP gene are extensively methylated. In addition, we describe the existence of polymorphism in the AFP and albumin genes of Sprague-Dawley rats.