RESUMO
The major focus of intrahepatic arterial (IHA) administration of adenoviruses (Ad) has been on safety. Currently, there is little published data on the biological responses to Ad when administered via this route. As part of a Phase I study, we evaluated biological responses to a replication-defective adenovirus encoding the p53 transgene (SCH 58500) when administered by hepatic arterial infusion to patients with primarily colorectal cancer metastatic to the liver. In analyzing biological responses to the Ad vector, we found that both total and neutralizing Ad antibodies increased weeks after SCH 58500 infusion. The fold increase in antibody titers was not dependent on SCH 58500 dosage. The proinflammatory cytokine interleukin-6 (IL-6) transiently peaked within 6 h of dosing. The cytokine sTNF-R2 showed elevation by 24 h post-treatment, and fold increases were directly related to SCH 58500 doses. Cytokines TNF-alpha, IL-1beta, and sTNF-R1 showed no increased levels over 24 h. Predose antibody levels did not appear to predict transduction, nor did serum Ad neutralizing factor (SNF). Delivery of SCH 58500 to tumor tissue occurred, though we found distribution more predominantly in liver tissues, as opposed to tumors. RT-PCR showed significantly higher expression levels (P<0.0001, ANOVA) for adenovirus type 2 and 5 receptor (CAR) in liver tissues, suggesting a correlation with transduction. Evidence of tumor-specific apoptotic activity was provided by laser scanning cytometry, which determined a coincidence of elevated nuclear p53 protein expression with apoptosis in patient tissue. IHA administration of a replication defective adenovirus is a feasible mode of delivery, allowing for exogenous transfer of the p53 gene into target tissues, with evidence of functional p53. Limited and transient inflammatory responses to the drug occurred, but pre-existing immunity to Ad did not preclude SCH 58500 delivery.
Assuntos
Adenoviridae/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Genes p53/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Neoplasias Hepáticas/secundário , Adulto , Idoso , Análise de Variância , Anticorpos Antivirais/sangue , Apoptose/efeitos dos fármacos , Neoplasias Colorretais/imunologia , Citocinas/sangue , Primers do DNA , Feminino , Artéria Hepática , Humanos , Infusões Intra-Arteriais , Citometria de Varredura a Laser , Masculino , Pessoa de Meia-Idade , Receptores Virais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In vitro experiments have demonstrated intercellular trafficking of the VP22 tegument protein of herpes simplex virus type 1 from infected cells to neighboring cells, which internalize VP22 and transport it to the nucleus. VP22 also can mediate intercellular transport of fusion proteins, providing a strategy for increasing the distribution of therapeutic proteins in gene therapy. Intercellular trafficking of the p53 tumor suppressor protein was demonstrated in vitro using a plasmid expressing full-length p53 fused in-frame to full-length VP22. The p53-VP22 chimeric protein induced apoptosis both in transfected tumor cells and in neighboring cells, resulting in a widespread cytotoxic effect. To evaluate the anti-tumor activity of p53-VP22 in vivo, we constructed recombinant adenoviruses expressing either wild-type p53 (FTCB) or a p53-VP22 fusion protein (FVCB) and compared their effects in p53-resistant tumor cells. In vitro, treatment of tumor cells with FVCB resulted in enhanced p53-specific apoptosis compared to treatment with equivalent doses of FTCB. However, in normal cells there was no difference in the dose-related cytotoxicity of FVCB compared to that of FTCB. In vivo, treatment of established tumors with FVCB was more effective than equivalent doses of FTCB. The dose-response curve to FVCB was flatter than that to FTCB; maximal antitumor responses could be achieved using FVCB at doses 1 log lower than those obtained with FTCB. Increased antitumor efficacy was correlated with increased distribution of p53 protein in FVCB-treated tumors. This study is the first demonstration that VP22 can enhance the in vivo distribution of therapeutic proteins and improve efficacy in gene therapy.
Assuntos
Adenovírus Humanos , Vetores Genéticos , Herpesvirus Humano 1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Apoptose , Células COS , Caspase 9 , Caspases/metabolismo , Chlorocebus aethiops , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Ativação Enzimática , Expressão Gênica , Humanos , Neoplasias Experimentais/fisiopatologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteínas Estruturais Virais/genéticaRESUMO
TP53 is the most commonly altered tumor-suppressor gene in cancer and is currently being tested in Phase II/III gene replacement trials. Many tumors contain wild-type TP53 sequence with elevated MDM2 protein levels, targeting p53 for degradation. These tumors are more refractory to treatment with exogenous wild-type p53. Here we generate a recombinant adenovirus expressing a p53 variant, rAd-p53 (d 13-19), that is deleted for the amino acid sequence necessary for MDM2 binding (amino acids 13-19). We compared the apoptotic activity of rAd-p53 (d 13-19) with that of a recombinant adenovirus expressing wild-type p53 (rAd-p53) in cell lines that differ in endogenous p53 status. rAd-p53 (d 13-19) caused higher levels of apoptosis in p53 wild-type tumor lines compared with wild-type p53 treatment, as measured by annexin V-FITC staining. In p53-altered tumor lines, rAd-p53 (d 13-19) showed apoptotic activity similar to that seen with wild-type p53 treatment. In normal cells, no increase in cytopathicity was detected with rAd-p53 (d 13-19) compared with wild-type p53 treatment. This variant protein displayed synergy with chemotherapeutic agents to inhibit proliferation of ovarian and breast cell lines. The p53 variant showed greater antitumor activity in an established p53 wild-type tumor compared with treatment with wild-type p53. The p53 variant represents a means of expanding TP53 gene therapy to tumors that are resistant to p53 treatment due to the cellular responses to wild-type p53.
Assuntos
Apoptose , Neoplasias da Mama/terapia , Genes p53 , Neoplasias Ovarianas/terapia , Proteína Supressora de Tumor p53/genética , Adenoviridae/genética , Animais , Caspase 9 , Caspases/metabolismo , Divisão Celular/fisiologia , Cisplatino/administração & dosagem , Terapia Combinada , Feminino , Variação Genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Paclitaxel/administração & dosagem , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/biossínteseRESUMO
Reports suggest a role of calpains in degradation of wild-type p53, which may regulate p53 induction of apoptosis. A calpain inhibitor, n-acetyl-leu-leu-norleucinal (calpain inhibitor 1), was assessed for ability to enhance p53-dependent apoptosis in human tumor cell lines with endogenous wild-type p53 and in altered p53 cell lines with the replacement of wild-type p53 by a recombinant adenovirus (rAd-p53). Calpain inhibitor 1 treatment resulted in increased levels of activated p53, increased p21 protein, and activation of caspases. Cell lines with wild-type, but not mutated or null, p53 status arrested in G0/G1 and were sensitive to calpain inhibitor-induced apoptosis. Regardless of endogenous p53 status, calpain inhibitor treatment combined with rAd-p53, but not empty vector virus, enhanced apoptosis in tumor cell lines. These results demonstrate p53-dependent apoptosis induced by a calpain inhibitor and further suggest a role for calpains in the regulation of p53 activity and induction of apoptotic pathways.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Glicoproteínas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Infecções por Adenoviridae/metabolismo , Western Blotting , Carcinoma Hepatocelular , Caspases/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/análise , Relação Dose-Resposta a Droga , Fase G1/fisiologia , Humanos , NF-kappa B/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fase de Repouso do Ciclo Celular/fisiologia , Fator de Transcrição AP-1/metabolismo , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologia , Proteína Supressora de Tumor p53/análiseRESUMO
Polyomavirus was originally isolated by Ludwick Gross from a mixture that also contained a murine retrovirus. A possible pathogenic interaction between polyomavirus and an endogenous mouse retrovirus locus (mtv-7) in polyomavirus-induced cancer has also been reported. To study potential interactive effects of polyomavirus (Py) and Moloney murine leukemia retrovirus (M-MuLV), newborn Balb/c and NIH Swiss mice were infected with high titer wild-type Py (A2 strain) and M-MuLV. Dramatically stunted growth (runting) occurred in 100% of the doubly inoculated mice, while much lower frequency of runting occurred in animals infected with Py alone and not at all with M-MuLV-infected mice. In situ hybridization for Py DNA showed ongoing Py replication and inflammation in kidneys (atypical of most mice singly infected by Py) of runted doubly inoculated mice. In addition, high Py viral replication continued well past the usual acute stage termination. M-MuLV replication was also initially inhibited in bone marrow by simultaneous Py infection. No M-MuLV replication was seen in singly or doubly infected mouse kidneys. Runting was very rapid, observable within 2 days after co-infection, arguing against an adaptive or antigen-specific immunological mechanism. One possibility was that a cytokine-driven acute response mechanism was involved. Supporting this view, RNAse protection assays for various cytokine RNAs showed that several were specifically elevated in kidneys of doubly infected mice. Three patterns were observed: (1) IL-6 was elevated in doubly infected mice early after infection (7 days), but it declined at later times (19 days); (2) IFN-gamma, IL-1 beta, and IL-10 were elevated at both early and late times; and (3) TNF-alpha, IL-12p40, and possibly TNF-beta were elevated only at late times. While the cytokines in the third category might be indicative of infiltrating inflammatory cells, it seems possible that cytokines in the first or second categories might be involved in establishing runting and ongoing polyoma DNA replication in the doubly infected mice.
Assuntos
Vírus JC/fisiologia , Leucemia Experimental/virologia , Vírus da Leucemia Murina de Moloney/fisiologia , Infecções por Polyomavirus/virologia , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/virologia , Animais , Animais Recém-Nascidos , Antígenos Transformantes de Poliomavirus/análise , Peso Corporal , Medula Óssea/virologia , Citocinas/biossíntese , DNA Viral/análise , Vírus JC/imunologia , Rim/crescimento & desenvolvimento , Rim/metabolismo , Rim/virologia , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/biossíntese , Replicação ViralRESUMO
Parotid tumor agent was an early name for polyomavirus due to its ability to induce tumors (myoepitheliomas originating from the myoepithelial glandular tissues) in mice inoculated with polyomavirus (Py) as neonates. It has long been thought that these tumors directly follow productive infection by Py in epithelial cells of the salivary gland, allowing subsequent cellular genetic changes leading to tumor formation. Curiously, the ability of salivary glandular tissue to support Py infection has not been experimentally established. Although Southern analysis for Py DNA has shown virus DNA to be present in whole salivary glands during acute infection, salivary glands are composed of various cell types (myoepithelial glandular cells called serous and mucous cells, fibrocollagenous cells, and interstitial cells), not all of which become transformed. We now use in situ hybridization for Py DNA along with immunohistological and immunohistochemical analyses to show that salivary gland serous and mucous cells are nonpermissive for acute Py infection in Balb/C and C3H mice, but are Py infected, as shown by T-ag expression. Salivary gland fibroblasts and interstitial cells, however, were permissive for Py replication. In addition, isoproterenol and tannin, which induce hypertrophy and hyperplasia of the secretory cells of adult male mice salivary glands, did not make these cells permissive to Py replication.
Assuntos
Polyomavirus/crescimento & desenvolvimento , Glândulas Salivares/microbiologia , Animais , Animais Recém-Nascidos , Antígenos Virais de Tumores/isolamento & purificação , Tecido Conjuntivo/microbiologia , Epitélio/microbiologia , Fibroblastos/microbiologia , Hipertrofia , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Glândula Parótida/metabolismo , Glândula Parótida/microbiologia , Glândula Parótida/patologia , Glândulas Salivares/metabolismo , Glândulas Salivares/patologia , Glândula Submandibular/metabolismo , Glândula Submandibular/microbiologia , Glândula Submandibular/patologia , Distribuição Tecidual , Replicação ViralRESUMO
Previous observations led us to propose that ongoing cellular differentiation, rather then proliferation, may be needed for high-level polyomavirus (Py) replication in permissive organs in vivo. We further tested this proposal by using the C57BL/6J-cpk/cpk mouse model for the autosomal recessive form of polycystic kidney disease (PKD) because both cellular proliferation and differentiation continue into the adult kidney in separate and distinct regions of the kidneys, whereas normal adult kidneys are quiescent and nonpermissive to Py. Adult PKD mice inoculated with Py were assayed for Py DNA replication by in situ hybridization and Southern analysis and for viral gene expression by immunofluorescence 5 days postinfection. The proliferation of collecting duct tubules of PKD kidneys was confirmed by in situ autoradiography for tritiated thymidine incorporation but were observed to be nonpermissive for Py gene expression or replication. Normal differentiated collecting ducts, however, are capable of supporting Py replication in non-PKD runted mice (Rochford et al., 1992). Py DNA, large T-Ag, and VP1, however, were detected in the nonproliferating distal and proximal tubules of the PKD parenchyma. The parenchymal tissues appear to be differentiating in a compensatory response to cyst growth. These results further support the view that in vivo Py replication may require ongoing cellular differentiation rather then mitosis.
Assuntos
Proteínas do Capsídeo , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Túbulos Renais Coletores/microbiologia , Doenças Renais Policísticas/microbiologia , Polyomavirus/fisiologia , Replicação Viral/fisiologia , Animais , Animais Recém-Nascidos , Capsídeo/análise , DNA/análise , DNA Viral/análise , Células Epiteliais , Epitélio/química , Túbulos Renais Coletores/química , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas Virais/análise , Doenças Renais Policísticas/patologiaRESUMO
Kidneys of newborn (but not adult) mice are normally high permissive for polyomavirus (Py) infection and readily establish persistent infections. We have proposed that ongoing cellular differentiation, which occurs in newborn mice, may be necessary for a high level of in vivo Py replication (R. Rochford, J. P. Moreno, M. L. Peake, and L. P. Villarreal, J. Virol. 66:3287-3297, 1992). This cellular differentiation requirement may also be necessary for the reactivation of a persistent Py kidney infection and could provide an alternative to the accepted view that reactivation results from immunosuppression. To examine this proposal, the ability of adult BALB/c mouse kidneys to support primary acute Py infection or to reactivate previously established persistent Py infections after kidney-specific damage was investigated. Kidney damage was induced by both chemical (glycerol, cisplatin, or methotrexate) and mechanical (through renal artery clamping to produce unilateral renal ischemia) treatments. We also examined the effects of epidermal growth factor (EGF), which enhances the rate of kidney regeneration, on Py replication. Using histopathologic techniques, in situ hybridization for Py DNA, and immunofluorescence for Py VP1 production, we established that both chemical damage and damage through renal artery clamping of adult kidneys promoted high levels of primary Py replication in these normally nonpermissive cells. This damage also promoted the efficient reactivation of Py replication from persistently infected kidneys, in the absence of immunosuppression. EGF treatment significantly increased acute Py replication and also reactivation in damaged kidneys. These results support the view that ongoing cellular division and differentiation may be needed both for high levels of acute Py replication and for reactivation of persistent infections in vivo.