Reactivation of latent Epstein-Barr virus by methotrexate: a potential contributor to methotrexate-associated lymphomas.

Background: Patients with rheumatoid arthritis or polymyositis treated with methotrexate (MTX) develop Epstein-Barr virus (EBV)-positive lymphomas more frequently than patients treated with other, equally immunosuppressive regimens. Here we determined whether MTX, in contrast to other commonly used medications for rheumatoid arthritis or polymyositis, is unique in its ability to induce the release of infectious EBV from latently infected cells.

Methods: The effect of MTX and other immunosuppressant drugs on EBV replication in vitro was assessed using latently infected EBV-positive lymphoblastoid and gastric carcinoma cell lines.

Phosphorylation- and Skp1-independent in vitro ubiquitination of E2F1 by multiple ROC-cullin ligases.

Ubiquitin-dependent proteolysis plays a critical role in the control of many cellular processes and is mediated by a cascade of enzymes involving ubiquitin activating (El), conjugating (E2), and ligating (E3) activities. Cullin 1/CDC53 functions as an E3 ligase by interacting with RING finger protein ROC1 and recruiting phosphorylated substrate. We report here that E2F1 transcription factor can be ubiquitinated in vitro and in vivo by multiple ROC-cullin ligases.

Association with cullin partners protects ROC proteins from proteasome-dependent degradation.

Cullin 1/CDC53 represents a multigene family and has been linked to the ubiquitin-mediated proteolysis of several different proteins. We recently identified two closely related RING finger proteins, ROC1 and ROC2, that share considerable sequence similarity to an APC subunit, APC11, and demonstrated ROC1 as an essential subunit of CUL1 and CDC53 ubiquitin ligases. We report here that the expression of ROC1, ROC2 and APC11 genes are induced by mitogens and remain constant during the cell cycle.

Identification of human cytomegalovirus target sequences in the human immunodeficiency virus long terminal repeat. Potential role of IE2-86 binding to sequences between -120 and -20 in promoter transactivation.

Objective: Because of the important medical consequences of human cytomegalovirus (HCMV) infection in human immunodeficiency virus (HIV)-infected individuals, we wanted to understand the molecular interactions that occur during co-infection. Specifically, in this study, we wanted to identify the transactivating target sequences on the HIV long terminal repeat (LTR) that responded to HCMV infection.

Study Design/methods: In this study, we transfected the HIV-LTR into human fibroblasts and then mapped the regulation of this promoter following HCMV infection and co-transfection with the HCMV immediate-early (IE) gene product IE2-86.

A role for transcription factor NF-kappa B in intestinal inflammation.

Nuclear factor (NF) kappa B is a transcription factor that controls the transcription of a variety of cellular genes regulating the inflammatory response. Many proinflammatory cytokines are transcriptionally regulated by NF-kappa B, and their increased expression has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Even though it seemed clear that the increase in proinflammatory cytokine production in IBD is crucial for the initiation and perpetuation of chronic intestinal inflammation, the elements governing this dysregulation of enhanced cytokine production remained unclear.

An enhanced EBNA1 variant with reduced IR3 domain for long-term episomal maintenance and transgene expression of oriP-based plasmids in human cells.

The latent replication of oriP-based plasmids in human cells depends on the viral oriP-binding transactivator EBNA1. In this report, the effect of the internal repeat 3 (IR3 or GlyAla repeat) domain of EBNA1 on long-term maintenance and transgene expression of OriP-based plasmids was examined in dividing human cells. To assess the potential contribution of different isoforms of EBNA1 specifically, the long-term stability of oriP-based plasmids was determined after stable transfection of various CMV-driven EBNA1 genes in EBV-negative human B cells.

Physicians' recommendations for colon cancer screening in women. Too much of a good thing?

Background: Expert groups support periodic colorectal cancer (CRC) screening for persons aged 50 and older but not for persons younger than 50. We were interested in community primary care physicians' recommendations to women for fecal occult blood tests (FOBT), flexible sigmoidoscopy (SIG), and colonoscopy (COL).

Methods: In a mailed survey of 1,292 community primary care physicians in North Carolina, we queried physicians regarding their recommendations to women for CRC screening.

Mammalian artificial chromosomes as tools for gene therapy.

Mammalian artificial chromosomes (MACs) represent powerful tools for human gene therapy and animal transgenesis. First-generation linear genomic human artificial chromosomes (HACs) and circular chimeric genomic/viral mouse artificial episomal chromosomes (MAECs) have been developed. HACs have been shuttled from human into mouse embryonal stem cells and human trans-chromosomic mice have been generated.

DNA signals for G2 checkpoint response in diploid human fibroblasts.

DNA (deoxyribonucleic acid) signals that induce the G2 checkpoint response were examined using proliferative secondary cultures of diploid human fibroblasts. Treatments that generated DNA double-strand breaks (DSBs) directly were effective inducers of checkpoint response, generally producing >80% inhibition of mitosis (G2 delay) and the kinase activity of M-phase-promoting factor within 2 h of treatment. Effective inducers of G2 checkpoint response included gamma-irradiation and the cancer chemotherapeutic drugs, bleomycin and etoposide.

Chromosomal instability is correlated with telomere erosion and inactivation of G2 checkpoint function in human fibroblasts expressing human papillomavirus type 16 E6 oncoprotein.

Cell cycle checkpoints and tumor suppressor gene functions appear to be required for the maintenance of a stable genome in proliferating cells. In this study chromosomal destabilization was monitored in relation to telomere structure, lifespan control and G2 checkpoint function. Replicative senescence was inactivated in secondary cultures of human skin fibroblasts by expressing the human papillomavirus type 16 (HPV-16) E6 oncoprotein to inactivate p53.

Expression of telomerase in normal and malignant rat hepatic epithelia.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA repeats onto the ends of chromosomes. More than 85% of human cancers express telomerase activity and a large proportion of human hepatocellular carcinomas are positive. To study the role of telomerase expression in rat hepatocarcinogenesis, telomerase activity was assayed in various rat tissues and in two types of liver epithelial cells: hepatocytes and hepatic epithelial stem-like cells.

NF-kappa B as a target for anti-inflammatory gene therapy: suppression of inflammatory responses in monocytic and stromal cells by stable gene transfer of I kappa B alpha cDNA.

One of the most challenging issues of anti-inflammatory gene therapy is the complexity of inflammatory pathways. Transcription factor NF-kappa B plays a pivotal role in activation of multiple inflammatory molecules, and therefore represents the logical target for intervention. We evaluated the feasibility of suppressing the inflammatory responses in different cell lines through specific inhibition of NF-kappa B by gene transfer of I kappa B alpha, the naturally occurring intracellular inhibitor of NF-kappa B.

Race and mammography use in two North Carolina counties.

Objectives: This study investigated racial differences in mammography use and their association with physicians' recommendations and other factors.

Methods: The study used 1988 survey data for 948 women 50 years of age and older from the New Hanover Breast Cancer Screening Program. Racial differences in terms of physician recommendation, personal characteristics, health characteristics, and attitudes toward breast cancer and mammography were examined.

The Epstein-Barr virus (EBV) DNA polymerase accessory protein, BMRF1, activates the essential downstream component of the EBV oriLyt.

The EBV DNA polymerase accessory protein, BMRF1, is an essential component of the viral DNA polymerase and is required for lytic EBV replication. In addition to its polymerase accessory protein function, we have recently reported that BMRF1 is a transcriptional activator, inducing expression of the essential oriLyt promoter, BHLF1. Here we have precisely mapped the BMRF1-response element in the BHLF1 promoter.

Engineering a mini-herpesvirus as a general strategy to transduce up to 180 kb of functional self-replicating human mini-chromosomes.

The engineering of therapeutic human artificial episomal chromosomes, HAECs, requires the development of strategies to deliver large functional self-replicating extrachromosomal DNA in target cells. Members of the herpesviral family are among the largest episomal double-stranded DNA viruses. As model systems of this family of endemic infectious agents, vectors derived from the human herpes 4 Epstein-Barr virus (EBV) were constructed which transferred up to 180 kb of DNA packaged as infectious virions.

Characterization of the mouse transforming growth factor alpha gene: its expression during eyelid development and in waved 1 tissues.

The spontaneous mouse waved 1 (wa1) mutation is allelic with the transforming growth factor alpha (TGF-alpha) gene and produces phenotypes similar to those of TGF-alpha knockout mice. Here, we show that TGF-alpha mRNA and protein levels are measurable in wa1 tissues but reduced 5- to 30-fold relative to wild type. Because the wa1-coding sequence is identical to that of the normal mRNA, wa1 is not a null mutation.

Sequence analysis of the human DNA flanking sites of human immunodeficiency virus type 1 integration.

Human immunodeficiency virus type 1 (HIV-1) tagged with the Escherichia coli supF gene has been used to clone integrated HIV-1 proviruses. Sequence analysis of the 600 to 800 bp of human DNA adjacent to 29 clones revealed a propensity for HIV-1 to integrate near the Alu class of human repetitive elements.

Visualization of the unwinding of long DNA chains by the herpes simplex virus type 1 UL9 protein and ICP8.

UL9 protein and ICP8 encoded by the herpes simplex virus type 1 (HSV-1) were shown to catalyze a highly active, non-origin-dependent unwinding of DNA. UL9 protein, the HSV-1 origin binding protein, as a modest helicase activity that is greatly stimulated by the HSV-1 single strand (ss) binding protein, ICP8. Here, electron microscopy has been applied to examine the mechanics of this reaction.

Transcriptional and post-transcriptional induction of the TGFalpha gene in transformed rat liver epithelial cells.

Although TGFalpha mRNA and protein are frequently elevated in neoplastic cells, neither the level at which deregulation occurs nor the mechanism(s) responsible have been well characterized. As a first step, we examined the induction of TGFalpha mRNA in two series of clonally-derived rat liver epithelial cell lines that were transformed either by exposure to chemical carcinogen or stable transfection of activated Ha-ras. We found that steady-state levels of TGFalpha mRNA in both series of transformed lines were induced 25- to 50-fold over those in the respective normal parental cells.

The herpes simplex virus type 1 origin-binding protein carries out origin specific DNA unwinding and forms stem-loop structures.

The UL9 protein of herpes simplex virus type 1 (HSV-1) binds specifically to the HSV-1 oriS and oriL origins of replication, and is a DNA helicase and DNA-dependent NTPase. In this study electron microscopy was used to investigate the binding of UL9 protein to DNA fragments containing oriS. In the absence of ATP, UL9 protein was observed to bind specifically to oriS as a dimer or pair of dimers, which bent the DNA by 35 degrees +/- 15 degrees and 86 degrees +/- 38 degrees respectively, and the DNA was deduced to make a straight line path through the protein complex.

Differential activation of the Ras/extracellular-signal-regulated protein kinase pathway is responsible for the biological consequences induced by the Axl receptor tyrosine kinase.

To understand the mechanism of Axl signaling, we have initiated studies to delineate downstream components in interleukin-3-dependent 32D cells by using a chimeric receptor containing the recombinant epidermal growth factor (EGF) receptor extracellular and transmembrane domains and the Axl kinase domain (EAK [for EGF receptor-Axl kinase]). We have previously shown that upon exogenous EGF stimulation, 32D-EAK cells are capable of proliferation in the absence of interleukin-3. With this system, we determined that EAK-induced cell survival and mitogenesis are dependent upon the Ras/extracellular-signal-regulated protein kinase (ERK) cascade.

Therapeutic gene delivery in human B-lymphoblastoid cells by engineered non-transforming infectious Epstein-Barr virus.

The B-lymphotrophic human herpes Epstein-Barr virus (EBV) is a 160-kilobase double-stranded DNA episomal virus carried in a persistent asymptomatic state by more than 90% of the worldwide adult population. We engineered a helper-dependent mini-EBV, with the minimal cis-EBV elements for episomal replication, viral amplification and packaging, for use as a gene delivery system. The therapeutic potential of this system was established by stably transducing B-lymphoblastoid cells from a Fanconi anaemia group C (FA-C) patient with a mini-EBV constitutively expressing the normal FACC cDNA and showing in vitro correction of the FA phenotype.

A kinase-deficient splice variant of the human JAK3 is expressed in hematopoietic and epithelial cancer cells.

Signal transduction of cytokine receptors is mediated by the JAK family of tyrosine kinases. Recently, the kinase partners for the interleukin (IL)-2 receptor have been identified as JAK1 and JAK3. In this study, we report the identification of splice variants that may modulate JAK3 signaling.

Human cytomegalovirus upregulates NF-kappa B activity by transactivating the NF-kappa B p105/p50 and p65 promoters.

During human cytomegalovirus (HCMV) infection, a series of regulated events take place following virus binding and entry into the cell, including the upregulation of cellular transcription factors, such as NF-kappa B, which play an essential role in the viral life cycle. We show here that NF-kappa B message is induced during HCMV infection and that the induction is biphasic, suggesting an initial induction at immediate-early (IE) times and a second round of induction at early times. This hypothesis is supported by experiments using cyclohexamide, which showed that the first tier of induction was drug insensitive, while the second tier was drug sensitive.