Sathyamangalam Swaminathan

p300/cAMP-responsive Element-binding Protein Interactions with Ets-1 and Ets-2 in the Transcriptional Activation of the Human Stromelysin Promoter

In this paper we show that transcription factors Ets-1 and Ets-2 recruit transcription adapter proteins p300 and CBP (cAMP-responsive element-binding protein) during the transcriptional activation of the human stromelysin promoter, which contains palindromic Ets-binding sites. Ets-2 and p300/CBP exist as a complexin vivo. Two regions of p300/CBP between amino acids (a.a.) 328 and 596 and a.a. 1678 and 2370 independently can interact with Ets-1 and Ets-2 in vitro and in vivo. Both these regions of p300/CBP bind to the transactivation domain of Ets-2, whereas the C-terminal region binds only to the DNA binding domain of Ets-2. The N- and the C-terminal regions of CBP (a.a. 1–1097 and 1678–2442, respectively) which lack histone acetylation activity independently are capable of coactivating Ets-2. Other Ets family transcription factors failed to cooperate with p300/CBP in stimulating the stromelysin promoter. The LXXLL sequence, reported to be important in receptor-coactivator interactions, does not appear to play a role in the interaction of Ets-2 with p300/CBP. Previous studies have shown that the stimulation of transcriptional activation activity of Ets-2 requires phosphorylation of threonine 72 by the Ras/mitogen-activated protein kinase signaling pathway. We show that mutation of this site does not affect its capacity to bind to and to cooperate with p300/CBP.

Abrogation of a mitotic checkpoint by E2 proteins from oncogenic human papillomaviruses correlates with increased turnover of the p53 tumor suppressor protein

Human papillomavirus (HPV) E2 and E1 proteins are required for the replication of viral genomes in vivo. We have examined the effects of increasing the level of E2 on viral and cellular replication using recombinant adenoviruses. Infection of cells which maintain HPV 31 DNA episomally with E2 recombinant adenoviruses resulted in a 5‐fold increase in genome copy number as well as an S phase arrest allowing for the continued replication of cellular DNA. Similar effects on cell cycle progression were seen following infection of normal human foreskin keratinocytes, the natural host cell. The DNA content of these cells increased beyond 4N indicating that multiple rounds of replication had occurred without an intervening mitotic event. In addition, increased cyclin A and E associated kinase activity was observed, while no change was detected in cyclin B associated kinase activity or in the activation state of cdc2 kinase. Interestingly, the levels of the p53 tumor suppresser protein were dramatically reduced through a post‐transcriptional mechanism following infection. These data suggest a role for E2 in regulating viral and cellular replication by abrogation of a mitotic checkpoint, which is, at least in part, controlled by p53.

Adenovirus-mediated tissue-targeted expression of the HSVtk gene for the treatment of breast cancer

In an effort to develop a genetic therapy for the treatment of breast cancer, we constructed adenoviral vectors containing either the beta-galactosidase (β-gal) reporter gene or the herpes simplex thymidine kinase (HSVtk) suicide gene driven by breast tissue-specific promoters. We utilized upstream regulatory sequences from either the human alpha-lactalbumin (hALA) gene, or the ovine beta-lactoglobulin (oBLG) gene in these vector constructs to target expression of heterologous genes transcriptionally to breast cancer cells both in vitro and in vivo. Data derived from breast tissue-specific reporter vectors in vitro demonstrate that expression from the hALA and oBLG promoters are indeed specific for breast cells (T47D, MCF-7, ZR75–1) when compared with non-breast cells (U2OS, HeLa). Moreover, these vectors displayed tumor cell specificity when compared with the normal MCF-10A breast cell line. These vectors also displayed breast tissue specificity when injected systemically (i.v.) into lactating Balb/c mice, which suggests that these promoters maintain their tissue- specific expression pattern within the context of the adenoviral genome in vivo. Tumors, derived from T47D human breast cancer cells, were established in nude mice and injected with either the tissue-specific reporter or suicide vectors. Results from tumors injected (i.t.) with reporter adenoviruses demonstrate that these promoters are active in T47D cells when grown as established tumors and we observed a marked regression of tumors injected with suicide vectors and treated systemically with gancyclovir (150 mg/kg/day) when compared with control animals. Moreover, mouse survival was prolonged after 35 days in mice undergoing therapy with the suicide vectors in conjunction with gancyclovir when compared with the control animals. These data suggest that the transcriptionally targeted hALA or oBLG driven expression of the HSVtk gene may be a feasible therapy for the treatment of human breast cancer.

Regulation of Adenovirus E2 Transcription Unit

The ability of viruses to take over the synthetic machinery of the host cells they invade for their own survival affords an ideal setting to look at the intricacies of ongoing regulation of macromolecular events and enables one to address specific questions concerning gene regulation. Genetic studies with animal viruses have provided detailed insights into the structure and function of the viral genome and the gene products it encodes. Viral mutants provide a means of probing the genetic and biochemical phenomena occurring in infected cells. Adenoviruses are members of DNA tumor virus family and are widespread in nature. Although about 40 different serotypes of human adenoviruses have been identified, adenovirus (Ad2) and Ad5 are the most well studied. The relatively small size (36 kilo bases) of its genome and the ease with which it can be grown to high titers in cultured cells have made the adenoviruses particularly attractive for experimentation. Early promoters of human adenoviruses provide excellent model systems to study transcriptional regulation. In adenovirus-infected human cells, six early viral promoters are coordinately regulated, and their efficient transcription is dependant on the large E1A protein which consists of 289 amino acids (Flint and Shenk 1989). The upstream DNA sequence elements that are required for basal E1A-independent transcription of these promoters have been identified. A number of cellular transcription factors that interact with these elements in a sequence-dependent fashion have been identified and cloned.

Regulation of Cellular Genes in a Chromosomal Context by the Retinoblastoma Tumor Suppressor Protein

ABSTRACT The retinoblastoma tumor suppressor gene product (pRb) is involved in controlling cell cycle progression from G1 into S. pRb functions, in part, by regulating the activities of several transcription factors, making pRb involved in the transcriptional control of cellular genes. Transient-transfection assays have implicated pRb in the transcription of several genes, including c-fos, the interleukin-6 gene, c-myc,cdc-2, c-neu, and the transforming growth factor β2 gene. However, these assays place the promoter in an artificial context and exclude the effects of far 5′ upstream regions and chromosomal architecture on gene transcription. In these experiments, we have studied the role of pRb in the control of cell cycle-related genes within a chromosomal context and within the context of the G1 phase of the cell cycle. We have used adenovirus vectors to overexpress pRb in human osteosarcoma cells and breast cells synchronized in early G1. By RNase protection assays, we have assayed the effects of this virus-produced pRb on gene expression in these cells. These results indicate that pRb is involved in the transcriptional downregulation of the E2F-1, E2F-2, dihydrofolate reductase, thymidine kinase, c-myc, proliferating-cell nuclear antigen, p107, and p21/Cip1 genes. However, it has no effect on the transcription of the E2F-3, E2F-4, E2F-5, DP-1, DP-2, or p16/Ink4 genes. The results are consistent with the notion that pRb controls the transcription of genes involved in S-phase promotion. They also suggest that pRb negatively regulates the transcription of two of the transcription factors whose activity it also represses, E2F-1 and E2F-2, and that it plays a role in downregulating the immediate-early gene response to serum stimulation.

Translation Control by Adenovirus Virus-Associated RNA I

Adenoviruses were first isolated by Rowe and co-workers from human adenoid tissues—hence the name adenoviruses. These are nonenveloped DNA viruses, composed of only DNA and proteins, and are widespread in nature. Forty-one of the 93 strains identified so far are of human origin, while the rest have been isolated from monkeys, dogs, cattle, rodents, and birds.1–3 Clinical studies have shown that these viruses can cause a variety of diseases in humans, most of which involve the respiratory tract, the eye, and the gastrointestinal tract. For the most part, these infections lead to self-limited illnesses or latent persistent infections and are usually followed by complete recovery. Adenoviruses can maintain inapparent infections within host animals for months or even years. Lymphoid cells may serve as reservoirs for these persistent infections. Several members of this group can also induce malignant tumors in newborn rodents and therefore adenoviruses are classified as members of the DNA tumor virus group. All the members of this group tested so far have been shown to transform rodent cells in culture.4 Despite their tumorigenic potential in experimental models, these viruses have not been shown to be tumorigenic in humans. Adenoviruses can be grown to high titers in a variety of cultured mammalian cells. Human cells of epithelial origin are best suited for growing human adenoviruses. Because of the small size of their genome, the ease with which they can be grown in rapidly growing cultured cells, and their ability to transform rodent cells in vitro, adenoviruses have served as important model systems to study the regulation of eukaryotic gene expression and virus-host-cell interactions.