Assessment of an altered E1B promoter on the specificity and potency of triple-regulated conditionally replicating adenoviruses: implications for the generation of ideal m-CRAs.

Although previous studies modified two components of conditionally replicating adenoviruses (CRAs), which selectively replicate in and kill cancer cells, the most accurate ways to achieve increased cancer specificity (that is, safety) without reducing the anticancer (that is, therapeutic) effects are unknown. Here, we generated two types of survivin-responsive m-CRAs (Surv.m-CRAs), Surv.m-CRA-CMVp and Surv.m-CRA-OCp, which use two and three different mechanisms to target cancer, that is, early region 1A (E1A) regulated by the survivin promoter and mutated E1BΔ55K regulated by the ubiquitously active cytomegalovirus promoter and cancer/tissue-specific osteocalcin promoter, respectively, and carefully examined their safety and anticancer effects. Endogenous osteocalcin mRNA was expressed and further enhanced by vitamin D(3) in all osteosarcoma and prostate cancer cell lines and human osteoblasts, but not in human fibroblasts. The osteocalcin promoter activity was weak even with vitamin D(3) treatment in these osteocalcin-expressing cancers, leading to low E1BΔ55K expression after Surv.m-CRA-OCp infection. Nevertheless, Surv.m-CRA-OCp had significantly increased cancer specificity without reduced anticancer effects in both in vitro and in vivo experiments. The unexpected but favorable fact that strong activity of an altered E1B promoter is unnecessary indicates that the majority of cancer/tissue-specific promoters may be used to generate ideal m-CRAs and will advance the development of m-CRA-based cancer therapies.

Retrovirus-mediated in vivo gene transfer in the replicating liver using recombinant hepatocyte growth factor without liver injury or partial hepatectomy.

Retrovirus-mediated gene delivery into hepatocytes in vivo provides long-term gene expression, which is of great importance for treating most genetic and metabolic disorders. However, clinical application has not been realized because of the requirement for prior 70% partial hepatectomy or chemical (toxic) liver injury to initiate hepatocyte replication at the time of retroviral gene transduction. In this paper, we describe a novel gene delivery system that uses recombinant hepatocyte growth factor (rHGF) prior to retrovirus-mediated in vivo gene transfer in the liver without partial hepatectomy or liver injury. A single retroviral infusion through the portal vein following five systemic injections (via the tail vein) of 100 microg/kg rHGF resulted in a 10.4% 5-bromo-2'-deoxyuridine (BrdU) labeling index (BLI) and 0.14% retroviral gene transduction efficiency (RGTE) in hepatocytes, which were 6.3- and 12.9-fold higher than those of controls, respectively. Modest additional increases in BLI and RGTE (13.4% and 0.22%, respectively) were seen after five systemic injections of 500 microg/kg rHGF. The correlation between BLI and RGTE was statistically confirmed regardless of treatment. When rats received multiple retroviral infusions through a cannulated portal vein following five portal injections of 100 microg/kg rHGF, RGTE was dramatically increased (1.3%) and in some areas of the liver exceeded more than 10%. There was no evidence of liver injury in any animal. This approach has great potential for clinical application in terms of avoiding invasive procedures or liver injury.

Regenerating livers of old rats contain high levels of C/EBPalpha that correlate with altered expression of cell cycle associated proteins.

The nuclear transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha) is expressed at high levels in the liver and inhibits growth in cultured cells. We have tested the correlation between C/EBPalpha levels, cell cycle proteins and hepatocyte proliferation in old and young animals as an in vivo model system in which the proliferative response to partial hepatectomy (PH) has been shown to be reduced and delayed in old animals. Here we present evidence that the expression of C/EBPalpha in old rats (24 months) differs from its expression in young animals (6-10 months) during liver regeneration. Induction of proliferating cell nuclear antigen (PCNA), a marker of DNA synthesis, occurs at 24 h after PH in young rats but is delayed and reduced in old animals. Induction of the mitotic-specific protein, cdc2 p34, is 3-4-fold less in regenerating liver of old rats than in the liver of young animals, confirming the reduced proliferative response in old animals. In young rats, the normal regenerative response involves a reduction of 3-4-fold in the levels of C/EBPalpha protein at 3-24 h. In old animals, C/EBPalpha is not reduced within 24 h after PH, but a decrease of C/EBPalpha protein levels can be detected at 72 h after PH. Induction of C/EBPbeta, another member of the C/EBP family, is delayed in old animals. Changes in the expression of C/EBP proteins are accompanied by alteration of the CDK inhibitor, p21, which is also decreased in young rats after PH, but in old animals remains unchanged. High levels of p21 protein in older animals correlate with the lack of cdk2 activation. We suggest that the failure to reduce the amount of C/EBPalpha and p21 is a critical event in the dysregulation of hepatocyte proliferation in old animals following PH.

Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma.

Recombinant adenoviral mediated delivery of suicide and cytokine genes has been investigated as a treatment for hepatic metastases of colon carcinoma in mice. Liver tumors were established by intrahepatic implantation of a poorly immunogenic colon carcinoma cell line (MCA-26), which is syngeneic in BALB/c mice. Intratumoral transfer of the herpes simplex virus type 1 thymidine kinase (HSV-tk) and the murine interleukin (mIL)-2 genes resulted in substantial hepatic tumor regression, induced an effective systemic antitumoral immunity in the host and prolonged the median survival time of the treated animals from 22 to 35 days. The antitumoral immunity declined gradually, which led to tumor recurrence over time. A recombinant adenovirus expressing the mIL-12 gene was constructed and tested in the MCA-26 tumor model. Intratumoral administration of this cytokine vector alone increased significantly survival time of the animals with 25% of the treated animals still living over 70 days. These data indicate that local expression of IL-12 may also be an attractive treatment strategy for metastatic colon carcinoma.

Neonatal cholestasis in two siblings: a variant of Dubin-Johnson syndrome?

OBJECTIVE: Two Japanese brothers with neonatal cholestasis associated with pigment granules in the hepatocytes and hepatosteatosis were evaluated for the possible role of hepatosteatosis in the Dubin-Johnson syndrome. METHODOLOGY AND RESULTS: The morphology of pigment accumulation and the laboratory data in these cases were examined. The elevation of urinary coproporphrin isomer I to more than 90% and the presence cholestasis resembled that in the Dubin-Johnson syndrome, but the hypertriglyceridaemia ( > 1.13 mmol/L as triolein) and the hepatosteatosis differed. Both infants were thought to have familial hypertriglyceridaemia. However, this diagnosis was difficult to confirm in the absence of data on the normal values of apolipoprotein and lipoprotein isomer for infants. CONCLUSIONS: A neonatal variant of the Dubin-Johnson syndrome may account for the unusual findings in these infants.