Efficacy of herpes simplex virus thymidine kinase in combination with cytokine gene therapy in an experimental metastatic breast cancer model.

Immunotherapy in combination with suicide gene therapy for breast cancer was tested using a metastatic animal model. Subcutaneous injection of the nonimmunogenic breast cancer cell line 4T1 in BALB/C mice gave rise to tumors in 100% of mice with both micrometastases and macrometastases in the lung. We used the herpes simplex virus thymidine kinase (HSV-TK) gene along with the cytokine genes granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) to determine their effect on tumor regression and inhibition of lung metastasis. Adenoviral (AV) vectors carrying these transgenes, in separate constructs, were used in this study. Intratumoral administration of AV-TK followed by 10 days of ganciclovir treatment resulted in a delay in tumor growth and, in some cases, in a low to moderate reduction in tumor volume. Inclusion of either GM-CSF or IL-2 gene with HSV-TK resulted in a slightly greater reduction in tumor volume, although these data were not significantly different from those obtained for TK treatment alone. However, when both cytokine genes were combined with TK, a substantial reduction in tumor growth was observed compared with HSV-TK alone (P < .02). Tumor weight data also exhibited superior efficacy of TK/GM-CSF/IL-2 treatment when compared with animals treated with TK gene only (P < .01). More importantly, TK/GM-CSF/IL-2 combination gene therapy induced a significant reduction in lung metastasis compared with any other treatment groups in the 4T1 model (P < .001 between TK GM-CSF/IL-2 versus TK only). Surgical excision of primary tumors after TK/GM-CSF/IL-2 plus ganciclovir treatment resulted in anti-metastatic activity that was similar to that observed for animals in which no surgery was performed. Survival analysis showed a significant difference between animals treated with AV-TK/GM-CSF/IL-2 and animals treated with TK only at 35 days after virus injection (P < .01). Immunophenotypic data suggest infiltration of lymphocytes within the tumor microenvironment in TK- and cytokine gene-treated animals. Thus, the overall data presented here demonstrate that TK gene therapy in combination with GM-CSF and IL-2 gene-mediated immunotherapy strategies have important implications in the treatment of breast cancer.

Inhibition of apoptosis in human immunodeficiency virus-infected cells enhances virus production and facilitates persistent infection.

Apoptosis is one of several mechanisms by which human immunodeficiency virus type 1 (HIV-1) exerts its cytopathic effects. CD4+ Jurkat T-cell lines overexpressing the adenovirus E1B 19K protein, a potent inhibitor of apoptosis, were used to examine the consequences of inhibition of apoptosis during acute and chronic HIV-1 infections. E1B 19K protein expression inhibited HIV-induced apoptosis, enhanced virus production, and established high levels of persistent viral infection. One E1B 19K-expressing line appeared to undergo HIV-induced death via a nonapoptotic mechanism, illustrating that HIV infection results in lymphocyte depletion through multiple pathways. Increased virus production associated with sustained cell viability suggests that therapeutic approaches involving inhibition of HIV-induced programmed cell death may be problematic.

NF-kappa B-dependent and -independent pathways of HIV activation in a chronically infected T cell line.

J delta K cells were isolated as a chronically infected survivor cell line, following infection of Jurkat CD4+ T cells with dl-NF, a mutated strain of human immunodeficiency virus type 1 (HIV-1) containing a deletion of the long terminal repeat (LTR) NF-kappa B sites. J delta K cells exhibited very low levels of constitutive HIV production. HIV-1 expression was activated from J delta K cells by treatment with phorbol myristate acetate (PMA), sodium butyrate (NaB), or hexamethylene bisacetamide (HMBA), but not tumor necrosis factor alpha (TNF-alpha), confirming the role of NF-kappa B in mediating TNF-alpha induction of HIV transcription. The strong induction of HIV expression by NaB or HMBA in J delta K cells clearly demonstrates the existence of NF-kappa B-independent mechanisms of HIV activation in chronically infected cells. J delta K cells may provide a useful model for characterizing NF-kappa B-independent transcriptional activation of the HIV LTR.

Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels.

The rep gene of adeno-associated virus type 2 (AAV) encodes four overlapping Rep proteins that are involved in gene regulation and replication of the virus. We studied here the regulation of mRNA transcribed from the AAV p5 and p19 promoters, using transient expression in human 293 cells followed by Northern (RNA) blot analysis of the mRNA. The p5 transcript encodes the larger Rep proteins, Rep78 and Rep68, while the p19 transcript encodes the smaller proteins, Rep52 and Rep40. A plasmid (pNTC3) containing the entire AAV genome with an amber mutation in the rep gene accumulated higher levels of p5 and p19 mRNA than a plasmid containing the wild-type AAV genome. Addition of increasing amounts of the wild-type rep gene in trans from a heterologous promoter inhibited p5 and p19 mRNA accumulation from pNTC3, indicating that the levels of both transcripts were decreased by the Rep proteins. Cotransfections with plasmids producing individual wild-type Rep proteins in trans showed that p5 and p19 mRNA accumulation was inhibited 5- to 10-fold by Rep78 and Rep68 and 2- to 3-fold by Rep52 and Rep40. Analysis of carboxyl-terminal truncation mutants of Rep78 showed that the ability of Rep78 to decrease p5 and p19 mRNA levels was lost when 159 or more amino acids were deleted. Rep78 and Rep68 mutants deleted for the methionine at residue 225 showed decreased abilities to down-regulate both p5 and p19 transcript levels, while mutants containing a substitution of glycine for the methionine resembled the wild-type Rep78. A Rep78 protein with a mutation in the putative nucleoside triphosphate binding site inhibited expression from p5 but not from p19, suggesting that the regulation of p5 transcript levels by Rep78 and Rep68 differs from that of p19. A deletion analysis of AAV cis sequences revealed that an intact terminal repeat was not required for negative regulation of p5 and p19 transcript levels and that the regulation of p19 mRNA levels by Rep78 did not require the presence of the p5 promoter.

Site-directed mutagenesis of cysteine residues of hepatitis B surface antigen. Analysis of two single mutants and the double mutant.

The structure of hepatitis B surface antigen (HBsAg) is mainly maintained by an intricate disulfide network responsible for most of its structural and antigenic properties. Characterization of three cysteine-replacement mutants of HBsAg has been performed by both structural and immunological methods. Replacement of Cys121 or Cys124 with serine results in mutant proteins that show diminished binding titres to both monoclonal antibodies and to a polyclonal serum, indicating that a structural change has taken place. Circular dichroism analysis shows that the substitution of either of these two residues also diminishes the helical content of the protein. However, the double mutant, in which both cysteine residues have been simultaneously changed, reverts the properties of the single mutations, and shows similar behaviour to the wild-type protein. Both the single and double cysteine mutants are efficiently glycosylated and secreted from Chinese hamster ovary cells and, in all cases, the mutant proteins assemble into spherical particles of similar buoyant density to both the wild-type and serum derived HBsAg.

Adenovirus containing a deletion of the early region 2A gene allows growth of adeno-associated virus with decreased efficiency.

Efficient growth of adeno-associated virus (AAV) requires helper functions provided by a coinfecting adenovirus or herpesvirus. Earlier studies using adenoviruses having temperature-sensitive lesions in the early region 2A gene (E2A) produced contradictory evidence regarding the role of the E2A 72-kDa DNA-binding protein (DBP) in allowing efficient AAV growth. These disparate results may reflect varying levels of residual function in the temperature-sensitive DBP. We examined this issue using an adenovirus type 5 mutant (Add/802) that fails to produce any detectable DBP or any fragment of it. Our experiments show that AAV can carry out a full growth cycle in the complete absence of DBP. However, AAV DNA replication and rep and capsid protein synthesis were reduced several fold and the yield of infectious AAV was reduced by an order of magnitude. This appears to reflect mainly decreased post-transcriptional expression of AAV rep and capsid protein genes.

Adeno-associated virus Rep protein inhibits human immunodeficiency virus type 1 production in human cells.

The adeno-associated virus (AAV) rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) required for AAV DNA replication and AAV gene regulation. In addition, the Rep proteins may have pleiotropic regulatory effects in heterologous systems, and in particular Rep78 may mediate a negative regulatory effect. We analyzed the effects of the AAV rep gene on human immunodeficiency virus type 1 (HIV-1) gene expression. The rep gene proteins of AAV type 2 (AAV2) inhibited the trans-activating ability of HIV-1. Constructs containing the AAV2 rep gene (pHIVrep) or a CAT gene (pBennCAT) expressed from the 5' HIV-1 long terminal repeat were inducible for Rep78 and Rep68 or CAT expression, respectively, when cotransfected with a plasmid containing the HIV-1 tat gene (pARtat). When equivalent amounts of pHIVrep and pBennCAT were cotransfected with increasing amounts of pARtat, expression of CAT activity was decreased. The pHIVrep construct was more inhibitory than plasmids expressing rep from the wild-type AAV2 p5 transcription promoter. rep expression from pHIVrep almost completely inhibited the replication of an HIV-1 proviral clone as measured by reverse transcriptase activity and p24 protein levels. Inhibition of HIV-1 production by Rep protein was also seen at the transcriptional level in that all HIV-1 transcripts were decreased when pHIVrep was present. The inhibitory effects of pHIVrep appear to be mediated primarily by Rep78 and perhaps Rep68. These results suggest that a trans-acting protein from a heterologous virus might be used to inhibit HIV-1 growth.

Naturally-occurring pituitary growth hormone is phosphorylated.

The results of this communication show that ovine growth hormone (oGH) contains organically-bound phosphorous. The phosphorous content of growth hormone, lot S-11, is 1:3 (mol/mol) and that of lot S-12 is 1:6 (mol/mol). Results of 31P NMR studies suggest that the phosphorous exists in two chemical forms: as a monophosphoryl ester and as a phosphodiester. Evidence is provided which demonstrates that growth hormone can be phosphorylated in vitro with the catalytic subunit of protein kinase.