acyclovir and Carcinoma

In this study, we aimed to construct an effective and safe oncolytic adenoviral vector for cancer treatment with gene therapy. First, the promoter of the catalytic subunit of human telomerase (hTERTp), adenovirus early region 1a gene (E1A) and thymidine kinase gene of human herpes virus type 1 (HSV-1-TK) were amplified by using PCR from genomic DNA of 293A cells and wild-type HSV-1 (wHSV-1). These specially-prepared elements were inserted into an adenoviral shuttle vector in the opposite and the same directions of left inverted terminal repeat (L-ITR), respectively, to construct pENTR-E1A-IRES-TK-hTERTp (pEITH) and pENTR-hTERTp-E1A-IRES-TK (pHEIT). LR reaction between adenoviral shuttle vectors (pEITH and pHEIT) and the backbone vector DEST was carried out to establish adenoviral expression vectors pAd-E1A-IRES-TK-hTERTp (pAd-EITH) and pAd-hTERTp-E1A-IRES-TK (pAd-HEIT). Recombinant adenovirus Ad-EITH and Ad-HEIT were produced by transfecting 293A cells and purified for the subsequent studies of titer measurement, replication capability with and without acyclovir (ACV) and antitumor ability with and without ganciclovir (GCV) to evaluate the biological characteristics. Adenoviral shuttle vectors pEITH and pHEIT and expression vectors pAd-EITH and pAd-HEIT were successfully constructed, and recombinant adenoviruses Ad-EITH and Ad-HEIT with high titer were produced. The results of replication and cytotoxicity assays showed that Ad-EITH and Ad-HEIT replicated in the hTERTp (+) human nasopharyngeal carcinoma cell line CNE and expressed the TK gene effectively leading to the death of tumor cells. In addition, there were still some Ad-HEIT particles replicating in the hTERTp (-) human osteosarcoma U-2OS cells and human lung HFL-1 fibroblasts compared to Ad-EITH which was hardly able to replicate in U-2OS and HFL-1 cells. In addition, we also observed an interesting phenomenon, that the replication of Ad-EITH could be inhibited by antiviral drug ACV on account of the expression of HSV-1-TK gene making Ad-EITH sensitive to ACV. In conclusion, a novel oncolytic adenoviral vector Ad-EITH was produced which can be used for cancer-specific and efficient viral replication, and its safety is potentially improved as replication can be inhibited by ACV in vitro.

Topics: Acyclovir; Adenoviridae; Adenovirus E1A Proteins; Antiviral Agents; Bone Neoplasms; Carcinoma; Cell Line, Tumor; Fibroblasts; Ganciclovir; Gene Amplification; Gene Expression; Genetic Therapy; Genetic Vectors; Herpesvirus 1, Human; Humans; Lung; Nasopharyngeal Neoplasms; Osteosarcoma; Promoter Regions, Genetic; Recombinant Proteins; Telomerase; Terminal Repeat Sequences; Thymidine Kinase; Virus Replication

Cells expressing herpes simplex virus (HSV) thymidine kinase (tk) are killed by ganciclovir (GCV). Adjacent cells without HSV-tk also die, a phenomenon known as the 'bystander effect'. However, there is no evidence that replication-competent HSV induces a bystander effect in the presence of GCV. Therefore, we investigated the bystander effect in HEp-2 cells infected with replication-competent, oncolytic HSV-1 mutants, hrR3 and HF10. In cells infected at a multiplicity of infection (MOI) of 3, GCV did not induce apoptosis. At low MOIs of 0.3 and 0.03, however, a number of adjacent, uninfected cells apoptosed following GCV treatment. Irrespective of GCV treatment, HEp-2 cells expressed minimal levels of connexin 43 (Cx43). However, Cx43 expression was enhanced by GCV in response to infection with HF10 at an MOI of 0.3, but not at an MOI of 3. Expression of other proteins involved in gap junctions, including Cx26 and Cx40, was not augmented under these conditions. The PKA and PI3K signal transduction pathways are likely involved in enhanced Cx43 expression as inhibitors of these pathways prevented Cx43 upregulation. These results suggest that infection with replication-competent HSV-1 induces the bystander effect in cells treated with GCV because of efficient intercellular transport of active GCV through abundant gap junctions.

Topics: Acyclovir; Animals; Antiviral Agents; Apoptosis; Bystander Effect; Carcinoma; Cell Line, Tumor; Chlorocebus aethiops; Connexin 26; Connexin 43; Connexins; Cyclic AMP-Dependent Protein Kinases; Ganciclovir; Herpesvirus 1, Human; Humans; Mutation; Oncolytic Virotherapy; Oncolytic Viruses; Phosphatidylinositol 3-Kinases; Thymidine Kinase; Transfection; Vero Cells; Virus Replication

The most extensively investigated strategy of suicide gene therapy for treatment of cancer is the transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene followed by administration of antiviral prodrugs such as acyclovir (ACV) and ganciclovir (GCV). The choice of the agent that can stimulate HSV-TK enzymatic activity is one of the determinants of the usefulness of this strategy. Previously, we found that a diterpenoid, scopadulciol (SDC), produced a significant increase in the active metabolite of ACV. This suggests that SDC may play a role in the HSV-TK/prodrug administration system.. The anticancer effect of SDC was evaluated in HSV-TK-expressing (TK+) cancer cells and nude mice bearing TK+ tumors. In vitro and in vivo enzyme assays were performed using TK+ cells and tumors. The phosphorylation of ACV monophosphate (ACV-MP) was measured in TK- cell lysates. The pharmacokinetics of prodrugs was evaluated by calculating area-under-the-concentration-time-curve values.. SDC stimulated HSV-TK activity in TK+ cells and tumors, and increased GCV-TP levels, while no effect of SDC was observed on the phosphorylation of ACV-MP to ACV-TP by cellular kinases. The SDC/prodrug combination altered the pharmacokinetics of the prodrugs. In accord with these findings, SDC enhanced significantly the cell-killing activity of prodrugs. The bystander effect was also significantly augmented by the combined treatment of ACV/GCV and SDC.. SDC was shown to be effective in the HSV-TK/prodrug administration system and improved the efficiency of the bystander effect of ACV and GCV. The findings will be considerably valuable with respect to the use of GCV in lower doses and less toxic ACV. This novel strategy of drug combination could provide benefit to HSV-TK/prodrug gene therapy.

Topics: Abietanes; Acyclovir; Animals; Antiviral Agents; Biological Availability; Carcinoma; Cell Line, Tumor; Female; Ganciclovir; Genes, Neoplasm; Genetic Therapy; Glioblastoma; HeLa Cells; Herpes Simplex; Herpesvirus 1, Human; Humans; Laryngeal Neoplasms; Mice; Mice, Nude; Molecular Structure; Plasmids; Prodrugs; Thymidine Kinase; Transfection; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

To develop a new toxic gene therapy using the tissue-specific human chorionic gonadotropin-beta (hCG-beta) promoter for testicular cancer. Although most patients presenting with disseminated testicular tumor are cured through the use of chemotherapy with or without surgery, those patients with relapse after initial therapy present a difficult clinical problem. The serum tumor marker hCG-beta is frequently elevated in patients with testicular cancer, and the pretreatment and post-treatment levels of serum hCG-beta are highly predictive of treatment outcome.. Human testicular embryonal carcinoma cell line, NEC 8, a human prostate cancer cell line, PC-3, and a human bladder cancer cell line, WH, were used in this study. A transient expression experiment was used to analyze the activity of a 729-bp hCG-beta promoter in all three cell lines. A recombinant adenovirus carrying thymidine kinase (Ad-hCG-beta-TK) under control of the hCG-beta promoter was generated. The tissue-specific activity of Ad-hCG-beta-TK was tested in vitro and in vivo.. The hCG-beta promoter had significantly greater activity in the hCG-beta-producing cell line (NEC 8) than in the non-hCG-beta-producing cell lines (PC-3 and WH). In vitro, Ad-hCG-beta-TK with acyclovir significantly inhibited NEC 8 growth but not PC-3 or WH cell growth. In vivo, Ad-hCG-beta-TK with acyclovir significantly inhibited NEC 8 subcutaneous tumor growth in nude mice.. In this study, we explored the possibility of developing a new therapeutic agent to target and induce the killing of testicular germ cell tumor selectively by using tissue-specific hCG-beta promoters.

Topics: Acyclovir; Adenoviridae; Animals; Biomarkers, Tumor; Carcinoma; Carcinoma, Embryonal; Cell Line, Tumor; Chorionic Gonadotropin, beta Subunit, Human; Enzyme Inhibitors; Genes, Transgenic, Suicide; Genetic Therapy; Genetic Vectors; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Prodrugs; Promoter Regions, Genetic; Prostatic Neoplasms; Recombinant Fusion Proteins; Salvage Therapy; Testicular Neoplasms; Thymidine Kinase; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

A 46-year-old man with HIV infection and AIDS presented with a large perianal ulcerated vegetative lesion that developed over a 1-year period. He had a past history of recurrent genital herpes infection, treated successfully each time with acyclovir. The perianal lesion developed while he was taking prophylactic acyclovir. Clinically, there were features suspicious of a carcinoma and a biopsy was reported as showing dysplasia. Therefore, the lesion was resected in its entirety. Histologically, there were prominent pseudo-epitheliomatous hyperplasia and chronic ulceration associated with herpesvirus infection. There was no evidence of dysplasia or malignancy. It is important to be aware of chronic vegetant herpesvirus infection, as clinical appearances are unusual and some methods of identification, such as smears or biopsy, may not be sufficient for diagnosis. Viral culture or PCR may need to be performed for a definite diagnosis to alleviate prolonged discomfort and avoid unnecessary radical surgery.

Topics: Acquired Immunodeficiency Syndrome; Acyclovir; AIDS-Related Opportunistic Infections; Antiviral Agents; Anus Neoplasms; Carcinoma; Diagnosis, Differential; Epithelial Cells; Fissure in Ano; Herpes Simplex; Humans; Hyperplasia; Immunocompromised Host; Male; Middle Aged; Papillomaviridae

To establish the expression of the herpes simplex virus thymidine kinase (HSV-TK) gene in tumor cells, we analyzed the promoter function of the SV40 promoter and the nucleotide sequence (CACGTG) to which Myc-Max heterodimers (Myc/Max) were capable of binding in four kinds of cell lines: COLO320 DM, A-431, KF, and Nakajima. When luciferase reporter plasmid under the control of SV40 promoter was transfected into tumor cells in vitro, a high level of luciferase activity was observed in all kinds of cell lines. However, by transfection of the luciferase gene promoted by the Myc/Max binding sequence, accelerated luciferase expression was observed in COLO320 DM and A-431 cells with high expression of c-myc, but not in KF and Nakajima cells, which showed low expression of c-myc. The repeated transfection of the liposome-conjugated HSV-TK gene regulated by the SV40 promoter and cultivation in 100 micrograms/ml of aciclovir for 5 days in vitro demonstrated growth inhibition for all four kinds of cell lines. However, cell toxicity was observed only in COLO320 DM and A-431 cells when the HSV-TK gene promoted by the Myc/Max binding sequence was introduced. (The survival rate to 100 micrograms/ml of aciclovir concentration in COLO320 DM, A-431, KF, and Nakajima cells was 59%, 53%, 74%, and 79%, respectively.) In vivo direct injection of the liposome-conjugated HSV-TK gene regulated by the SV40 promoter into established tumors and aciclovir administration for 10 days into the mice resulted in significant tumor volume reduction in three tested tumor cells. However, injection of the HSV-TK gene promoted by the Myc/Max binding sequence and aciclovir administration into mice could achieve significant tumor regression only in COLO320 DM and A-431 cells. These results suggest that gene therapy using the HSV-TK gene promoted by the Myc/Max binding sequence can be an attractive approach for treatment against tumor cells expressing high levels of c-myc.

Topics: Acyclovir; Animals; Antimetabolites; Base Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Basic-Leucine Zipper Transcription Factors; beta-Galactosidase; Binding Sites; Carcinoma; DNA-Binding Proteins; DNA, Recombinant; Drug Carriers; Gene Expression; Gene Transfer Techniques; Genes, Reporter; Genetic Therapy; Humans; Liposomes; Luciferases; Mice; Mice, Nude; Molecular Sequence Data; Neoplasms, Glandular and Epithelial; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Simplexvirus; Thymidine Kinase; Transcription Factors; Tumor Cells, Cultured

The most common causes of acute viral infections of the eye for which there are no effective antiviral drugs are the adenoviruses. Until recently, pathogenesis studies and antiviral drug testing for adenovirus-induced ocular disease were not practical because no animal model was available. However, new animal models for human adenovirus-induced ocular and respiratory infections have now made such studies possible. We assessed the in vitro and in vivo activity of ganciclovir against a genetically defined adenovirus (Ad5 wt 300) known to cause severe ocular disease. The 50% inhibitory dose (ID50) values were determined by plaque reduction assays in human cells. The ID50 values of 47 and 604 microM were determined for ganciclovir and acyclovir, respectively, against Ad5, and 26 and 152 microM, respectively against Ad8. Cotton rats were inoculated bilaterally with 10(5) plaque-forming units per eye and treated topically with ganciclovir (3%, 1%, or 0.3%) or placebo for 21 days. All inoculated eyes were culture positive on days 1-3 with increased infectivity titers, regardless of treatment. However, the incidence, duration, and titer of virus shed in eyes treated with 3% ganciclovir was reduced, and the antiadenovirus enzyme-linked immunosorbent assay titers in serum were lower in these animals. Although these differences were not statistically significant, the observed trend suggested that the highest ganciclovir dose had a suppressive effect on some disease parameters.

Topics: Acyclovir; Adenovirus Infections, Human; Adenoviruses, Human; Animals; Antibodies, Viral; Carcinoma; Enzyme-Linked Immunosorbent Assay; Eye Infections, Viral; Ganciclovir; Humans; Keratoconjunctivitis, Infectious; Lethal Dose 50; Lung Neoplasms; Ophthalmic Solutions; Sigmodontinae; Tumor Cells, Cultured; Virus Shedding