Faculty Bibliography

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro

We recently reported the cloning of a rearranged human oncogene following transfection of DNA from Kaposi's sarcoma into NIH 3T3 cells. To identify the protein(s) encoded in two novel mRNAs of 3.5 and 1.2 kb expressed in NIH 3T3 transformants, we constructed a cDNA library. One of the cDNA clones isolated (KS3) corresponded to the 1.2 kb mRNA and transformed NIH 3T3 cell when inserted into a mammalian expression vector. The 1152 nucleotide KS3 cDNA encodes a protein of 206 amino acids with significant homology to the growth factors basic FGF and acidic FGF. Expression of the KS3 product as a bacterial fusion protein or in COS cells allowed us to determine that both proteins had significant growth-promoting activity and that the COS cell protein was glycosylated. Thus one of the mRNAs transcribed from the KS oncogene encodes a growth factor that could transform cells by an autocrine mechanism and appears to represent a new member of the FGF family

By transfecting high molecular weight DNA from a Kaposi sarcoma lesion into murine NIH 3T3 cells, we have identified and molecularly cloned a set of human DNA sequences capable of inducing focus formation, growth in agar, and tumorigenicity in these cells. The human DNA sequences present in primary, secondary, and tertiary NIH 3T3 transformants encompass about 32 kilobases (kb) and contain four rearrangements with respect to normal human DNA and a portion of the c-fms protooncogene (FMS in human gene nomenclature). However, the minimal transforming region (6.6 kb) identified in our cloned DNA borders on the c-fms DNA region but does not contain c-fms coding sequences. The fms sequences are also not represented in the two transcripts (approximately equal to 1.2 and 3.5 kb) detected in NIH 3T3 transformants; however, they might provide elements regulating expression. Hybridization to several known oncogene probes and preliminary sequencing data indicate that we have identified a previously unrecognized 'activated' oncogene. Since the rearrangements present in our cloned DNA sequences are not detectable in the original Kaposi tumor DNA used for transfection, it is possible that this oncogene was generated during gene transfer

The RNA polymerase III-dependent transcription of B2 repeated sequences has been monitored during the transition from the quiescent to proliferative state in cultured rodent cells and after polyomavirus-induced transformation. The level of RNAs containing B2 sequences was found to be higher in both the proliferative state of normal cells and in polyomavirus-transformed cells. In both systems, nuclear run-off transcription assays indicated that high levels of B2 RNAs are due to an enhanced transcription rate. These results suggest the presence of a B2-specific RNA pol III transcription factor(s) whose activity is sensitive to cell cycle progression and oncogenic transformation

Polyoma virus (Py) large T-Antigen (LT) can promote the amplification of viral genomes integrated in the chromosomal DNA of rat fibroblasts, and this phenomenon requires the interaction of the LT protein with the viral origin of DNA replication. To compare the rate and the modality of selectable amplification events promoted by the Py LT with cellular-driven events, we constructed a double expression vector containing a murine dihydrofolate reductase (dhfr) cDNA and the bacterial chloramphenicol acetyl transferase (cat) gene controlled by the viral regulatory region. The plasmid was introduced into a rat cell line constitutively producing a temperature sensitive LT (cl 2), and clones were selected in low concentration of methotrexate (MTX). Three cl 2 transformants and one control cell line lacking LT were propagated at temperatures permissive (33 degrees C) and non permissive (39 degrees C) for LT function and, subsequently, challenged in one step with high MTX dosage at 39 degrees C. While the control line produced the same number of colonies irrespective of the temperature of propagation, the three LT positive lines yielded between 2 and greater than 100 times more colonies following propagation at permissive temperature, indicating that the presence of Py LT considerably increased the rate of amplification of integrated sequences linked to the viral origin of replication. In all cases the amplification event involved the exogenous and not the endogenous dhfr gene, and overexpression of the cat gene occurred as a result of co-amplification with the selectable dhfr sequences. Analysis of the structure of the amplified domain in the various resistant derivatives revealed that, in the presence of the viral protein, amplification occurred within the boundaries of the primary plasmid insert. In the absence of a functional LT protein, amplification both internal or involving adjacent host DNA were observed

We have cloned a human cDNA that complements the mutation of ts11, a temperature-sensitive (ts) mutant of the BHK hamster cell line that at the nonpermissive temperature is blocked in progression through the G1 phase of the cell growth cycle. After transfecting human chromosomal DNA into ts11 cells and selecting for cells that had acquired a non-ts phenotype, we screened a genomic library constructed in the EMBL3 lambda vector from a secondary non-ts transformant and isolated a recombinant phage containing human DNA sequences that were uniformly present in primary and secondary non-ts transformants. Genomic probes that recognized an mRNA of about 2 kilobases in human cells were used to isolate from a cDNA expression library two cDNA plasmids that could efficiently transform ts11 cells to a non-ts phenotype. Sequencing of one of these cDNAs revealed a single open reading frame, which could encode a 540 amino acid protein. The ts11 gene has at least two other homologs in human DNA and thus it appears to be part of a small gene/pseudogene family. Experiments with serum-synchronized cells indicate that the expression of the ts11 gene, which is necessary for G1 progression, is itself cell-cycle regulated, being induced in approximately mid-G1

AIDS (acquired immune deficiency syndrome) and ARC (AIDS-related complex) are associated with a spectrum of lymphoproliferative disorders ranging from lymphadenopathy syndrome (LAS), an apparently benign polyclonal lymphoid hyperplasia, to B cell non-Hodgkin's lymphoma (B-NHL), i.e., malignant, presumably monoclonal B cell proliferations. To gain insight into the process of lymphomagenesis in AIDS and to investigate a possible pathogenetic relationship between LAS and NHL, we investigated the clonality of the B or T lymphoid populations by Ig or T beta gene rearrangement analysis, the presence of rearrangements involving the c-myc oncogene locus, and the presence of human immunodeficiency virus (HIV) sequences in both LAS and B-NHL biopsies. Our data indicate that multiple clonal B cell expansions are present in a significant percentage of LAS (approximately 20%) and B-NHL (60%) biopsies. c-myc rearrangements/translocations are detectable in 9 of our 10 NHLs, but not in any of the LAS cases. However, only one of the B cell clones, identified by Ig gene rearrangements carries a c-myc gene rearrangement, suggesting that only one clone carries the genetic abnormality associated with malignant B cell lymphoma. Furthermore, the frequency of detection of c-myc rearrangements in AIDS-associated NHLs of both Burkitt and non-Burkitt type suggest that the biological alterations present in AIDS favor the development of lymphomas carrying activated c-myc oncogenes. Finally, our data show that HIV DNA sequences are not detectable in LAS nor in NHL B cell clones, suggesting that HIV does not play a direct role in NHL development. Taken together, these observations suggest a model of multistep lymphomagenesis in AIDS in which LAS would represent a predisposing condition to NHL. Immunosuppression and EBV infection present in LAS can favor the expansion of B cell clones, which in turn may increase the probability of occurrence of c-myc rearrangements leading to malignant transformation

The frequent occurrence of Kaposi's sarcoma (KS) in association with the acquired immune deficiency syndrome (AIDS) could be due to the fact that the etiological agent of this tumor is the same retrovirus causing AIDS, to another oncogenic virus frequently found in AIDS patients, or to the unmasking of the tumorigenic potential of KS cells by immunosuppression. We have therefore investigated the presence of DNA sequences homologous to the AIDS retrovirus, cytomegalovirus (CMV), and hepatitis B virus in 13 KS necropsies and biopsies from AIDS patients. All KS DNA samples were negative for AIDS retrovirus or hepatitis B DNA sequences. Two DNAs from necropsies contained CMV DNA, but the data suggested the presence of replicating CMV DNA due to generalized infection. We have also studied cell cultures derived from KS skin biopsies of AIDS patients. These cultures had a short lifetime in vitro and expressed some markers of endothelial cells. The cells were not tumorigenic in nude mice but contained a number of chromosomal rearrangements which were often monoclonal within the same culture. However, these abnormalities were different from culture to culture and even in cultures from the same biopsy. The presence of these chromosomal abnormalities seemed to correlate with the cell positivity for endothelial markers. Taken together these results indicate that neither the AIDS retrovirus, CMV, or hepatitis B virus is directly responsible for the altered growth of KS cells, that KS may be polyclonal even within the same lesion, and that KS cells have a tendency to karyotypic rearrangements

We have examined the effects of the E1a products of adenovirus types 5 and 12 on the expression of polyomavirus early and late promoters. In cotransfection experiments in HeLa cells, plasmids expressing the E1a region of adenovirus type 5 or 12 repressed both the early and late promoters of polyomavirus, and deletion analysis indicates that the polyomavirus enhancers were the target of the E1a repression. With mutants lacking enhancer sequences, the polyomavirus early promoter but not the late promoter was trans-activated by E1a. Chimeric mutant plasmids with deletions in the regulatory region that contained either the A enhancer or the B enhancer were repressed to the same extent, indicating that E1a can repress both elements. Polyomavirus variant plasmids with rearrangements in the regulatory region conferring activity in embryonal carcinoma stem cells were repressed by E1a as was the wild type, suggesting that the repressor function is quite general. We discuss a model in which the influence of E1a on the transcriptional activity of a gene is the sum of positive and negative effects on promoter and enhancer elements and discuss possible mechanisms of negative regulation of enhancer function