p300 provides a corepressor function by cooperating with YY1 and HDAC3 to repress c-Myc.

We showed earlier that p300/CBP plays an important role in G1 progression by negatively regulating c-Myc and thereby preventing premature G1 exit. Here, we have studied the mechanism by which p300 represses c-Myc and show that in quiescent cells p300 cooperates with histone deacetylase 3 (HDAC3) to repress transcription. p300 and HDAC3 are recruited to the upstream YY1-binding site of the c-Myc promoter resulting in chromatin deacetylation and repression of c-Myc transcription. Consistent with this, ablation of p300, YY1 or HDAC3 expression results in chromatin acetylation and induction of c-Myc. These three proteins exist as a complex in vivo and form a multiprotein complex with the YY1-binding site in vitro. The C-terminal region of p300 is both necessary and sufficient for the repression of c-Myc. These and other results suggest that in quiescent cells the C-terminal region of p300 provides corepressor function and facilitates the recruitment of p300 and HDAC3 to the YY1-binding site and represses the c-Myc promoter. This corepressor function of p300 prevents the inappropriate induction of c-Myc and S phase.

Relationship between E1A binding to cellular proteins, c-myc activation and S-phase induction.

We recently showed that p300/CREB-binding protein (CBP) plays an important role in maintaining cells in G0/G1 phase by keeping c-myc in a repressed state. Consistent with this, adenovirus E1A oncoprotein induces c-myc in a p300-dependent manner, and the c-myc induction is linked to S-phase induction. The induction of S phase by E1A is dependent on its binding to and inactivating several host proteins including p300/CBP. To determine whether there is a correlation between the host proteins binding to the N-terminal region of E1A, activation of c-myc and induction of S phase, we assayed the c-myc and S-phase induction in quiescent human cells by infecting them with Ad N-terminal E1A mutants with mutations that specifically affect binding to different chromatin-associated proteins including pRb, p300, p400 and p300/CBP-associated factor (PCAF). We show that the mutants that failed to bind to p300 or pRb were severely defective for c-myc and S-phase induction. The induction of c-myc and S phase was only moderately affected when E1A failed to bind to p400. Furthermore, analysis of the E1A mutants that fail to bind to p300, and both p300 and PCAF suggests that PCAF may also play a role in c-myc repression, and that the two chromatin-associated proteins may repress c-myc independently. In summary, these results suggest that c-myc deregulation by E1A through its interaction with these chromatin-associated proteins is an important step in the E1A-mediated cell cycle deregulation and possibly in cell transformation.

Kruppel-like factor 4 regulates laminin alpha 3A expression in mammary epithelial cells.

Laminin-5, the major extracellular matrix protein produced by mammary epithelial cells, is composed of three chains (designated alpha3A, beta3, and gamma2), each encoded by a separate gene. Laminin-5 is markedly down-regulated in breast cancer cells. Little is known about the regulation of laminin gene transcription in normal breast cells, nor about the mechanism underlying the down-regulation seen in cancer. In the present study, we cloned the promoter of the gene for the human laminin alpha3A chain (LAMA3A) and investigated its regulation in functionally normal MCF10A breast epithelial cells and several breast cancer cell lines. Using site-directed mutagenesis of promoter-reporter constructs in transient transfection assays in MCF10A cells, we find that two binding sites for Kruppel-like factor 4 (KLF4/GKLF/EZF) are required for expression driven by the LAMA3A promoter. Electrophoretic mobility shift assays reveal absence of KLF4 binding activity in extracts from T47D, MDA-MB 231, ZR75-1, MDA-MB 436, and MCF7 breast cancer cells. Transient transfection of a plasmid expressing KLF4 activates transcription from the LAMA3A promoter in breast cancer cells. A reporter vector containing duplicate KLF4-binding sites in its promoter is expressed at high levels in MCF10A cells but at negligible levels in breast cancer cells. Thus, KLF4 is required for LAMA3A expression and absence of laminin alpha3A in breast cancer cells appears, at least in part, attributable to the lack of KLF4 activity.

Adenovirus-mediated targeted expression of toxic genes to adrenocorticotropin-producing pituitary tumors using the proopiomelanocortin promoter.

Management of Cushing's disease remains challenging, despite advances in its diagnosis and treatment. Here, we describe a strategy for targeting the expression of toxic genes to ACTH-producing tumor cells using adenoviral vectors. The POMC promoter was used to express either a marker gene (beta-galactosidase) or a toxic gene [herpes simplex virus thymidine kinase (TK)]. In ACTH-producing AtT20 cells, infection with recombinant adenoviruses containing the POMC promoter (AdPOMCGal; AdPOMCTK) led to high-level gene expression. Stereotactic injection of AdPOMCGal into the rat pituitary resulted in localized expression of the beta-galactosidase transgene in corticotrope cells. Cytotoxicity studies were performed using the TK-containing vectors and treatment with ganciclovir. AdPOMCTK caused greater than 95% cytotoxicity of AtT20 cells at a viral dose (multiplicity of infection, 5 plaque-forming units/cell) that induced minimal toxicity using control viruses. No cellular toxicity was seen using a nonpituitary cell line (T47D breast tumor cells). AtT20 cells transplanted into nude mice induced features of Cushing's syndrome and were used as an in vivo model of ACTH-producing tumors. Injection of the AdPOMCTK virus caused significant regression of the transplanted AtT20 tumors. These studies suggest that the POMC promoter may provide a useful gene therapy strategy for the adjunctive treatment of pituitary tumors causing ACTH-dependent Cushing's syndrome.

Efficacy of adenoviral TNF alpha antisense is enhanced by a macrophage specific promoter.

Macrophage-derived TNF alpha is a critical mediator of inflammation and destruction in diseases such as rheumatoid arthritis and Crohn's disease. These studies were undertaken to develop an effective adenovirus-based strategy to specifically suppress TNF alpha in primary human macrophages. A variety of promoters and LTRs were evaluated for effective expression in the macrophage cell line RAW 264.7. The CMV promoter and the Visna LTR were the most strongly expressed and were therefore used to drive the expression of TNF alpha antisense fragments. In transient transfection assays, the antisense fragment terminating at the 3' end of the first exon (216 bp) was superior to the others (70 and 750 bp), when expressed under the control of either the CMV promoter or the Visna LTR. Adenoviral vectors expressing the 216 bp TNF alpha antisense fragment, controlled by the CMV promoter or the Visna LTR, were both effective at suppressing LPS-induced TNF alpha secretion by primary human macrophages. However, the Visna LTR was more effective not only at suppressing LPS-induced TNF alpha secretion, but also IL-6, which is highly sensitive to TNF alpha secretion. These results demonstrate that effective, specific, suppression of TNF alpha in macrophages is possible, employing a directed antisense approach and a promoter system that is highly efficient in human macrophages.

Antisense-mediated depletion of p300 in human cells leads to premature G1 exit and up-regulation of c-MYC.

The cAMP-response element-binding protein (CREB)-binding protein and p300 are two highly conserved transcriptional coactivators and histone acetyltransferases that integrate signals from diverse signal transduction pathways in the nucleus and also link chromatin remodeling with transcription. In this report, we have examined the role of p300 in the control of the G(1) phase of the cell cycle in nontransformed immortalized human breast epithelial cells (MCF10A) and fibroblasts (MSU) by using adenovirus vectors expressing p300-specific antisense sequences. Quiescent MCF10A and MSU cells expressing p300-specific antisense sequences synthesized p300 at much reduced levels and exited G(1) phase without serum stimulation. These cells also showed an increase in cyclin A and cyclin A- and E-associated kinase activities characteristic of S phase induction. Further analysis of the p300-depleted quiescent MCF10A cells revealed a 5-fold induction of c-MYC and a 2-fold induction of c-JUN. A direct target of c-MYC, CAD, which is required for DNA synthesis, was also found to be up-regulated, indicating that up-regulation of c-MYC functionally contributed to DNA synthesis. Furthermore, S phase induction in p300-depleted cells was reversed when antisense c-MYC was expressed in these cells, indicating that up-regulation of c-MYC may directly contribute to S phase induction. Adenovirus E1A also induced DNA synthesis and increased the levels of c-MYC and c-JUN in serum-starved MCF10A cells in a p300-dependent manner. Our results suggest an important role of p300 in cell cycle regulation at G(1) and raise the possibility that p300 may negatively regulate early response genes, including c-MYC and c-JUN, thereby preventing DNA synthesis in quiescent cells.

Regulation of IL-6 and IL-8 expression in rheumatoid arthritis synovial fibroblasts: the dominant role for NF-kappa B but not C/EBP beta or c-Jun.

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) produce IL-6 and IL-8, which contribute to inflammation and joint damage. The promoters of both cytokines possess binding sites for NF-kappaB, C/EBPbeta, and c-Jun, but the contribution of each to the regulation of IL-6 and IL-8 in RA FLS is unknown. We employed adenoviral-mediated gene delivery of a nondegradable IkappaBalpha, or dominant-negative versions of C/EBPbeta or c-Jun, to determine the contribution of each transcription factor to IL-6 and IL-8 expression. Inhibition of NF-kappaB activation significantly reduced the spontaneous and IL-1beta-induced secretion of IL-6 and IL-8 by RA FLS and the IL-1ss-induced production of IL-6 and IL-8 by human dermal fibroblasts. Inhibition of C/EBPbeta modestly reduced constitutive and IL-1beta-induced IL-6 by RA FLS, but not by human dermal fibroblasts, and had no effect on IL-8. Inhibition of c-Jun/AP-1 had no effect on the production of either IL-6 or IL-8. Employing gel shift assays, NF-kappaB, C/EBPbeta, and c-Jun were constitutively activated in RA FLS, but only NF-kappaB and c-Jun activity increased after IL-1beta. The reduction of cytokines by IkappaBalpha was mediated through inhibition of NF-kappaB activation, which resulted in decreased IL-6 and IL-8 mRNA. NF-kappaB was essential for IL-6 expression, because fibroblasts in which both NF-kappaB p50/p65 genes were deleted failed to express IL-6 in response to IL-1. These findings document the importance of NF-kappaB for the regulation of the constitutive and IL-1beta-stimulated expression of IL-6 and IL-8 by RA FLS and support the role of inhibition of NF-kappaB as a therapeutic goal in RA.

Regulation of TNF-alpha expression in normal macrophages: the role of C/EBPbeta.

C/EBPbeta is present in monocytes and macrophages, binds to the proximal region of the TNF-alpha promoter, and contributes to its regulation. This study was performed to characterize the ability of C/EBPbeta to regulate the TNF-alpha gene in myelomonocytic cells and primary macrophages. In transient transfection assays, overexpression of wild type C/EBPbeta resulted in a 3-4-fold activation of a 120 base pair TNF-alpha promoter-reporter construct, while overexpression of a dominant negative (DN) C/EBPbeta inhibited LPS-induced activation. In vitro monocyte-differentiated macrophages, infected with an adenoviral vector expressing the DN C/EBPbeta (AdDNC/EBPbeta) or the control Adbetagal, expressed their transgenes weakly, however expression was greatly enhanced in the presence of PMA. Infection with AdDNC/EBPbeta resulted in 60% suppression of LPS induced TNFalpha secretion compared to Adbetagal infection (P<0.001) in PMA-treated macrophages. Northern blot analysis demonstrated approximately a 40% reduction of the TNF-alpha mRNA in the presence of the DN C/EBPbeta, suggesting that the effect of the DN C/EBPbeta was at the transcriptional level. In contrast, AdDNC/EBPbeta infection did not result in inhibition of LPS-induced TNF-alpha secretion in the absence of PMA. Further, DN versions of both C/EBPbeta and c-Jun, but not NF-kappaB p65, suppressed PMA-induced TNF-alpha secretion in macrophages. These observations demonstrate that, C/EBPbeta and c-Jun contribute to the regulation of the TNF-alpha gene in normal macrophages following treatment with PMA.

Breast cancer-specific expression of the Candida albicans cytosine deaminase gene using a transcriptional targeting approach.

We constructed a series of adenoviral (Ad) vectors that express the Candida albicans cytosine deaminase (CD) suicide gene under the transcriptional control of either the human alpha-lactalbumin (ALA) or ovine beta-lactoglobulin (BLG) promoter (Ad.ALA.CD and Ad.BLG.CD, respectively). The Ad.ALA.CD and the Ad.BLG.CD vectors converted the prodrug 5-fluorocytosine (5-FC) to the toxic nucleotide analog 5-fluorouracil in a breast cancer cell-specific manner, with a conversion rate of 40% and 52% in T47D cells and 50% and 41% in MCF7 cells, respectively. No significant conversion (< or =3%) was observed in an immortalized nontumorigenic breast epithelial cell line (MCF10A) and a human osteosarcoma cell line (U2OS). Adenovirus vector-based prodrug conversion of the 5-FC in T47D and MCF7 in the presence of 1 mg/mL of 5-FC led to cytotoxicity that resulted in a nearly complete cell death (> or =90%) after 5 days, whereas MCF10A and U2OS cells remained resistant (< or =10%). Nude mice harboring T47D-derived breast tumors that were injected intratumorally (i.t.) with therapeutic adenovirus vectors at a dose of 2 x 10(8) plaque-forming units and treated systemically with 5-FC at a concentration of 500 mg/kg/day showed a marked reduction in tumor mass within 30 days when compared with animals that received vector alone. Animal survival was significantly prolonged after 72 days in mice treated with therapeutic vectors in conjunction with prodrug when compared with control animals. These preclinical data are sufficiently promising to warrant further studies of this transcriptional targeting approach to breast cancer treatment.

Stereotactic injection of adenoviral vectors that target gene expression to specific pituitary cell types: implications for gene therapy.

OBJECTIVE: Gene therapy is a potentially useful strategy for the treatment of pituitary adenomas or hormone deficiency disorders. We investigated the feasibility of targeting gene expression to specific pituitary cell types in vivo, using a combination of stereotactic injection and adenoviral vectors that carry pituitary-specific promoters. METHODS: Recombinant adenoviruses containing the human growth hormone promoter (AdGHGal) or the human glycoprotein hormone alpha-subunit promoter (AdalphaGal) were used to drive expression of the beta-galactosidase gene. The expression of beta-galactosidase activity in the pituitary was analyzed after the administration of recombinant adenoviruses via the peripheral vein or the carotid artery, or by stereotactic injection into the rat pituitary. Double-label histology was used to evaluate cell-type expression in the pituitary. RESULTS: Intravascular injection of AdGHGal or AdalphaGal failed to deliver the marker gene to the pituitary. However, direct stereotactic injection of recombinant adenoviral vectors into the pituitary achieved a high level of transgene expression. In addition, immunohistochemical staining revealed selective expression of the AdGHGal or AdalphaGal transgenes in pituitary cells that normally produce the respective hormones. CONCLUSION: These findings indicate that adenoviral vectors carrying pituitary gland-specific promoters may be useful for targeted gene therapy of pituitary diseases. However, because of low transduction after peripheral administration, stereotactic injection or local administration of viruses at the time of pituitary surgery is probably required for efficient gene expression.

Inhibition of laminin-5 production in breast epithelial cells by overexpression of p300.

The transcriptional coactivator p300 is essential for normal embryonic development and cellular differentiation. We have been studying the role of p300 in the transcription of a variety of genes, and we became interested in the role of this coactivator in the transcription of genes important in breast epithelial cell biology. From MCF-10A cells (spontaneously immortalized, nontransformed human breast epithelial cells), we developed cell lines that stably overexpress p300. These p300-overexpressing cells displayed reduced adhesion to culture dishes and were found to secrete an extracellular matrix deficient in laminin-5. Laminin-5 is the major extracellular matrix component produced by breast epithelium. Immunofluorescence studies, as well as experiments using normal matrix, confirmed that the decreased adhesion of p300-overexpressing cells is due to laminin-5-deficient extracellular matrix and not due to loss of laminin-5 receptors. Northern blots revealed markedly decreased levels of expression of two of the genes (designated LAMA3 and LAMC2) encoding the alpha3 and gamma2 chains of the laminin-5 heterotrimer in the cells that overexpress p300, whereas LAMB3 mRNA, encoding the third or beta3 chain of laminin-5, was not markedly reduced. Transient transfection experiments with a vector containing a murine LAMA3 promoter demonstrate that overexpressing p300 down-regulates the LAMA3 promoter. In summary, overexpression of p300 leads to down-regulation of laminin-5 production in breast epithelial cells, resulting in decreased adhesion.

Regulation of expression of N-methylpurine DNA glycosylase in human mammary epithelial cells: role of transcription factor AP-2.

The DNA repair enzyme, N-methylpurine DNA glyclosylase (MPG), is overexpressed in breast cancer as compared with its expression in normal breast epithelial cells. In an effort to determine the mechanism responsible for this difference in expression, we studied rates and regulation of transcription of the MPG gene in normal (HMEC), spontaneously immortalized (MCF10A), and malignant (T47D) mammary epithelial cells. Steady state levels of MPG mRNA are 3-4-fold greater in T47D cells than in MCF10A cells. Nuclear "run-off" transcription measurements revealed MPG transcription rates to be approximately 3-fold greater in the tumor cells than in normal cells. Characterization of the MPG promoter by deletion analysis and transient transfection experiments revealed that all basal promoter activity resided between nucleotides -227 and -81 upstream from the ATG translation start site. Constructs containing this region were expressed at 4-fold greater levels when transfected into malignant T47D cells (56 x baseline) than in MCF10A cells (14 x baseline). Computer database analysis of the region of nucleotides -227 to -81 revealed multiple overlapping Sp1 consensus binding sites and two overlapping consensus AP-2 binding sites located between bases -181 and -169. Electrophoretic mobility shift assays indicated that while Sp1 bound this region of the promoter, nuclear extracts from both cell types contained equal Sp1 binding activity. In contrast, AP-2 binding activity was significantly greater in T47D cells, and Western blots confirmed increased AP-2 protein levels in these cells. Cotransfection into MCF10A cells of the MPG promoter construct and an AP-2 expression plasmid increased MPG promoter activity 2.1-fold. Cotransfection of a dominant negative mutant of AP-2 into T47D cells reduced the extent of MPG promoter-driven transcription by 50%. To investigate the functional significance of the two overlapping AP-2 consensus binding sites, each site was mutated separately. Mutation of the upstream site decreased promoter activity by 15%, but mutation of the downstream site decreased promoter activity by 45% and abolished AP-2 binding to the promoter sequence. These data suggest that AP-2 is important in regulating MPG expression in breast cancer cells, and that the increased amount of AP-2 in these cells plays a major role in directing the increased expression of MPG.

Activation of the cyclin D1 gene by the E1A-associated protein p300 through AP-1 inhibits cellular apoptosis.

The adenovirus E1A protein interferes with regulators of apoptosis and growth by physically interacting with cell cycle regulatory proteins including the retinoblastoma tumor suppressor protein and the coactivator proteins p300/CBP (where CBP is the CREB-binding protein). The p300/CBP proteins occupy a pivotal role in regulating mitogenic signaling and apoptosis. The mechanisms by which cell cycle control genes are directly regulated by p300 remain to be determined. The cyclin D1 gene, which is overexpressed in many different tumor types, encodes a regulatory subunit of a holoenzyme that phosphorylates and inactivates PRB. In the present study E1A12S inhibited the cyclin D1 promoter via the amino-terminal p300/CBP binding domain in human choriocarcinoma JEG-3 cells. p300 induced cyclin D1 protein abundance, and p300, but not CBP, induced the cyclin D1 promoter. cyclin D1 or p300 overexpression inhibited apoptosis in JEG-3 cells. The CH3 region of p300, which was required for induction of cyclin D1, was also required for the inhibition of apoptosis. p300 activated the cyclin D1 promoter through an activator protein-1 (AP-1) site at -954 and was identified within a DNA-bound complex with c-Jun at the AP-1 site. Apoptosis rates of embryonic fibroblasts derived from mice homozygously deleted of the cyclin D1 gene (cyclin D1(-/-)) were increased compared with wild type control on several distinct matrices. p300 inhibited apoptosis in cyclin D1(+/+) fibroblasts but increased apoptosis in cyclin D1(-/-) cells. The anti-apoptotic function of cyclin D1, demonstrated by sub-G(1) analysis and annexin V staining, may contribute to its cellular transforming and cooperative oncogenic properties.

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 (beta-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.

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 complex in 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.

Recombinant adenovirus is an appropriate vector for endocytotic protein trafficking studies in cultured neurons.

Endocytosis of full-length beta-amyloid precursor protein (APP) from the plasma membrane contributes to beta-amyloid peptide (Abeta) secretion, and, hence, potentially contributes to the molecular pathogenesis of Alzheimer's disease. We recently have demonstrated that central neuronal APP is endocytosed in a common vesicular compartment with recycling synaptic vesicle integral membrane proteins, but is then sorted away from synaptic vesicles for retrograde transport to the neuronal soma. For this report, we explore whether recombinant adenovirus can be used to modulate APP expression in cultured central neurons to study APP processing by the endocytotic pathway in these cells. Using a replication-deficient recombinant adenovirus that expresses a lacZ reporter (Ad5/CMV-lacZ), we demonstrate high efficiency of transfection (30-35%) at low viral titer (10-20 MOI), with no significant neuronal toxicity or cytoarchitectural change. In addition, we demonstrate that infection with the control virus does not result in re-direction of endogenous neuronal APP from usual endocytotic pathways. We have prepared, using the same genomic background as the control virus, an adenoviral vector that expresses the neuronal isoform of human APP (Ad5/CMV-APP). Infection with Ad5/CMV-APP at 10-20 MOI results in significantly increased immunoreactivity for endocytosed APP with preservation of usual endocytotic trafficking. These results demonstrate that recombinant adenovirus at low titer is an appropriate and effective vector for protein trafficking/processing studies in cultured central neurons.

Targeted expression of toxic genes directed by pituitary hormone promoters: a potential strategy for adenovirus-mediated gene therapy of pituitary tumors.

Pituitary adenomas cause clinical manifestations because of mass effects and excess hormone production. This group of tumors represents a tractable target for gene therapy because they are rarely metastatic and because reductions in tumor size and function, in addition to those achieved after surgery, may be of clinical benefit. In this report we describe a strategy for targeting the expression of toxic genes to pituitary cells using adenoviral vectors. Pituitary hormone promoters (human GH or glycoprotein hormone alpha-subunit) were used to express either a marker gene [beta-galactosidase (beta-gal)] or a toxic gene [herpes simplex virus thymidine kinase (TK)]. In GH-producing GH3 cells and in alpha-subunit-producing pituitary tumor cell lines, recombinant adenoviruses containing either the alpha-subunit promoter (Ad alpha Gal; AdaTK) or the GH promoter (AdGHGal; AdGHTK) were expressed at-high levels. Using histological studies and assays for beta-gal activity, expression was shown to persist for at least 21 days, and it was relatively selective for pituitary cell lines. Cytotoxicity studies were performed using the TK-containing vectors and treatment with ganciclovir. Both AdGHTK and AdaTK caused greater than 95% cytotoxicity of GH3 and alphaT3 cells, respectively, at a viral dose (multiplicity of infection, 5 plaque-forming units/cell) that induced minimal toxicity using control viruses. Little cellular toxicity was seen using a nonpituitary cell line (T47D breast tumor cells). The AdGHTK virus also caused marked reduction in the size of GH3 cell tumors that were propagated in nude mice. These studies suggest that adenoviral vectors carrying human pituitary gland specific promoters may be useful for developing gene therapy strategies for the treatment of pituitary adenomas.

Regulation of cellular genes in a chromosomal context by the retinoblastoma tumor suppressor protein.

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 beta2 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.

Tumor necrosis factor alpha gene regulation: enhancement of C/EBPbeta-induced activation by c-Jun.

Tumor necrosis factor alpha (TNF alpha) is a key regulatory cytokine whose expression is controlled by a complex set of stimuli in a variety of cell types. Previously, we found that the monocyte/macrophage-enriched nuclear transcription factor C/EBPbeta played an important role in the regulation of the TNF alpha gene in myelomonocytic cells. Abundant evidence suggests that other transcription factors participate as well. Here we have analyzed interactions between C/EBPbeta and c-Jun, a component of the ubiquitously expressed AP-1 complex. In phorbol myristate acetate (PMA)-treated Jurkat T cells, which did not possess endogenous C/EBPbeta, expression of c-Jun by itself had relatively little effect on TNF alpha promoter activity. However, the combination of C/EBPbeta and c-Jun was synergistic, resulting in greater than 130-fold activation. This effect required both the leucine zipper and DNA binding domains, but not the transactivation domain, of c-Jun, plus the AP-1 binding site centered 102/103 bp upstream of the transcription start site in the TNF alpha promoter. To determine if C/EBPbeta and c-Jun might cooperate to regulate the cellular TNF alpha gene in myelomonocytic cells, U937 cells that possess endogenous C/EBPbeta and were stably transfected with either wild-type c-Jun or the transactivation domain deletion mutant (TAM-67) were examined. U937 cells expressing ectopic wild-type c-Jun or TAM-67 secreted over threefold more TNF alpha than the control line in response to PMA plus lipopolysaccharide. Transient transfection of the U937 cells expressing TAM-67 suggested that TAM-67 binding to the -106/-99-bp AP-1 binding site cooperated with endogenous C/EBPbeta in the activation of the -120 TNF alpha promoter-reporter. DNA binding assays using oligonucleotides derived from the TNF alpha promoter suggested that C/EBPbeta and c-Jun interact in vitro and that the interaction may be DNA dependent. Our data demonstrate that the TNF alpha gene is regulated by the interaction of the ubiquitous AP-1 complex protein c-Jun and the monocyte/macrophage-enriched transcription factor C/EBPbeta and that this interaction contributes to the expression of the cellular TNF alpha gene in myelomonocytic cells. This interaction was unique in that it did not require the c-Jun transactivation domain, providing new insight into the cell-type-specific regulation of the TNF alpha gene.

A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays.

At least three regions of the simian virus 40 small-t antigen (small-t) contribute to the protein's ability to enhance cellular transformation. As we showed previously for rat F111 cells, one region includes sequences from residues 97 to 103 that are involved in the binding and inhibition of protein phosphatase 2A. In the present study, the role of the protein phosphatase 2A binding region was confirmed in two additional small-t-dependent transformation systems. Second, small-t was found to provide a function previously identified as a large-T transformation domain. Mutations in residues 19 to 28 of large-T affected its transforming ability, but these mutations were complemented by a wild-type small-t. A third region of small-t was also required for efficient transformation. This region, the 42-47 region, is shared by large-T and small-t and contains a conserved HPDKGG hexapeptide. The 42-47 region function could be provided by either small-t or large-T in small-t-dependent systems. Mutations in the 42-47 region reduced the ability of small-t to transactivate the cyclin A promoter, of interest because small-t increased endogenous cyclin A mRNA levels in both human and monkey cells, as well as transactivating the promoter in transient assays.