Arming a replicating adenovirus with osteoprotegerin reduces the tumor burden in a murine model of osteolytic bone metastases of breast cancer.

Most patients with advanced breast cancer develop osteolytic bone metastases, which have numerous complications. Because current therapies are not curative, new treatments are needed. Conditionally replicating adenoviruses (CRAds) are anticancer agents designed to infect and lyse tumor cells. However, in spite of their promise as selective cancer therapeutics, replicating adenoviruses have shown limited efficacy in the clinical setting. We hypothesized that a CRAd armed with osteoprotegerin (OPG) would eradicate bone metastases of breast cancer both directly, by oncolysis, and indirectly, by inhibiting osteoclastic bone resorption, and thus reducing the tumor burden. We constructed an armed CRAd (Ad5-Δ24-sOPG-Fc-RGD) by replacing viral E3B genes with a fusion of the ligand-binding domains of OPG and the Fc portion of human IgG1. Conditional replication was conferred by a 24-base pair deletion within E1A (Δ24), which prevents the binding of E1A to the retinoblastoma tumor suppressor/cell cycle regulator protein and limits replication in normal cells. Enhanced infection of cells expressing low levels of the primary Ad5 receptor was conferred by incorporating an arginine-glycine-aspartic acid (RGD) peptide sequence into the fiber knob to mediate binding to α(v) integrins. After characterization of the armed CRAd, we demonstrated that infection of breast cancer cells by Ad5-Δ24-sOPG-Fc-RGD both killed the infected cells by oncolysis and inhibited the formation of osteoclasts in an in vitro co-culture model. In a murine model of osteolytic bone metastases of breast cancer, the CRAd armed with shortened OPG (sOPG)-Fc reduced tumor burden in the bone and inhibited osteoclast formation more effectively than an unarmed CRAd.

Employment of liver tissue slice analysis to assay hepatotoxicity linked to replicative and nonreplicative adenoviral agents.

Whereas virotherapy has emerged as a novel and promising approach for neoplastic diseases, appropriate model systems have hampered preclinical evaluation of candidate conditionally replicative adenovirus agents (CRAds) with respect to liver toxicity. This is due to the inability of human viral agents to cross species. We have recently shown the human liver tissue slice model to be a facile means to validate adenoviral replication. On this basis, we sought to determine whether our ex vivo liver tissue slice model could be used to assess CRAd-mediated liver toxicity. We analyzed and compared the toxicity of a conditionally replicative adenovirus (AdDelta24) to that of a replication incompetent adenovirus (Adnull [E1-]) in mouse and human liver tissue slices. To accomplish this, we examined the hepatic apoptosis expression profile by DNA microarray analyses, and compared these results to extracellular release of aminotransferase enzymes, along with direct evidence of apoptosis by caspase-3 immunhistochemical staining and TUNEL assays. Human and mouse liver tissue slices demonstrated a marked increase in extracellular release of aminotransferase enzymes on infection with AdDelta24 compared to Adnull. AdDelta24-mediated liver toxicity was further demonstrated by apoptosis induction, as detected by caspase-3 immunohistochemical staining, TUNEL assay and microarray analysis. In conclusion, concordance of CRAd-mediated apoptosis in both the human and the mouse liver tissue slice models was demonstrated, despite the limited replication ability of CRAds in mouse liver slices. The results of this study, defining the CRAd-mediated apoptosis gene expression profiles in human and mouse liver, may lay a foundation for preclinical liver toxicity analysis of CRAd agents.

Infectivity enhancement for adenoviral transduction of canine osteosarcoma cells.

The full realization of conditionally replicative adenoviruses (CRAds) for cancer therapy has been hampered by the limited knowledge of CRAd function in vivo and particularly in an immunocompetent host. To address this issue, we previously proposed a canine adenovirus type 2 (CAV2)-based CRAd for clinical evaluation in canine patients with osteosarcoma (OS). In this study, we evaluated infectivity-enhancement strategies to establish the foundation for designing a potent CAV2 CRAd with effective transduction capacity in dog osteosarcoma cells. The results indicate that the native CAV2 fiber-knob can mediate increased binding, and consequently gene transfer, in both canine osteosarcoma immortalized and primary cell lines relative to previously reported Ad5 infectivity-enhancement strategies. Gene delivery was further enhanced by incorporating a polylysine polypeptide onto the carboxy terminus of the CAV2 knob. This vector demonstrated improved gene delivery in osteosarcoma xenograft tumors. These data provide the rationale for generation of infectivity-enhanced syngeneic CAV2 CRAds for clinical evaluation in a dog osteosarcoma model.

A human adenoviral vector with a chimeric fiber from canine adenovirus type 1 results in novel expanded tropism for cancer gene therapy.

The development of novel therapeutic strategies is imperative for the treatment of advanced cancers like ovarian cancer and glioma, which are resistant to most traditional treatment modalities. In this regard, adenoviral (Ad) cancer gene therapy is a promising approach. However, the gene delivery efficiency of human serotype 5 recombinant adenoviruses (Ad5) in cancer gene therapy clinical trials to date has been limited, mainly due to the paucity of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human cancer cells. To circumvent CAR deficiency, Ad5 vectors have been retargeted by creating chimeric fibers possessing the knob domains of alternate human Ad serotypes. Recently, more radical modifications based on 'xenotype' knob switching with non-human adenovirus have been exploited. Herein, we present the characterization of a novel vector derived from a recombinant Ad5 vector containing the canine adenovirus serotype 1 (CAV-1) knob (Ad5Luc1-CK1), the tropism of which has not been previously described. We compared the function of this vector with our other chimeric viruses displaying the CAV-2 knob (Ad5Luc1-CK2) and Ad3 knob (Ad5/3Luc1). Our data demonstrate that the CAV-1 knob can alter Ad5 tropism through the use of a CAR-independent entry pathway distinct from that of both Ad5Luc1-CK2 and Ad5/3-Luc1. In fact, the gene transfer efficiency of this novel vector in ovarian cancer cell lines, and more importantly in patient ovarian cancer primary tissue slice samples, was superior relative to all other vectors applied in this study. Thus, CAV-1 knob xenotype gene transfer represents a viable means to achieve enhanced transduction of low-CAR tumors.

Evaluation of tumor-specific promoter activities in melanoma.

Gene therapy is a novel therapy for melanoma. To date, however, there is still no powerful tumor specific promoter (TSP) to restrict the transgene expression in melanoma cells. In order to define a useful TSP for targeting in the context of melanoma gene therapy, four promoters, the cyclooxygenase-2 (Cox-2), alpha-chemokine SDF-1 receptor (CXCR4), epithelial glycoprotein 2 (EGP-2), and survivin, were tested in both established melanoma cell lines and primary melanoma cells. We employed recombinant adenoviral vectors (reAds) each with a candidate TSP (the Cox-2, CXCR4, EGP-2, or survivin), a reporter luciferase gene, and a poly-A signal, all of which were inserted into the E1-deleted region. A reAdGL3Bcytomegalovirus (CMV), containing the CMV promoter and luciferase gene, was used as a positive control to normalize the luciferase activity. Luciferase activity was measured in multiple tumor cell lines and two primary melanoma cell cultures after infection with reAds. Human epithelial melanocytes, HEM, were used as normal control. In contrast to three other promoters, the survivin promoter exhibited the highest activities within both melanoma cell lines and primary melanoma cells, but not in HEMs. Additionally, the survivin promoter exhibited very low activities in major mouse organs including the liver, in vivo. EGP-2 is not active in melanoma; messenger RNA expressions were correlated to promoter activities both in melanoma cell lines and primary cell cultures. Thus, these data suggest that the survivin promoter achieved a 'tumor-on/liver-off' profile, and thus represents a potentially useful tumor-specific promoter with applications for transcriptional targeting of Ad vector-based cancer gene therapy or oncolysis to melanoma.

Combining high selectivity of replication with fiber chimerism for effective adenoviral oncolysis of CAR-negative melanoma cells.

Oncolytic adenoviruses constitute a new and promising tool for cancer treatment that has been rapidly translated into clinical trials. However, minimal or absent expression of the adenovirus serotype 5 (Ad5) receptor CAR (coxsackievirus and adenovirus receptor) on cancer cells represents a major limitation for Ad5-based oncolysis. Here, we report on the resistance of CAR-negative primary melanoma cells to cell killing by wild-type Ad5 (Ad5wt) even after high titer infection, thus underlining the need for tropism-modification of oncolytic adenoviruses. We engineered a new generation of oncolytic adenoviruses that exhibit both efficient target cell infection by swapping Ad5 fiber domains with those of Ad serotype 3, which binds to a receptor distinct from CAR, and targeted virus replication. Fiber chimerism resulted in efficient cytopathicity to primary melanoma cells, which was at least 10(4)-fold increased relative to Ad5wt. Since viral infectivity mediated by such modified viral capsids was not cell type-specific, it was pivotal to carefully restrict adenoviral replication to target cells. Towards this end, we replaced both E1A and E4 promoters of fiber chimeric viruses by tyrosinase enhancer/promoter constructs. The resulting viruses showed melanoma-specific expression of E1A and E4 and combined efficient virus replication and cell killing in melanoma cell lines and primary melanoma cells with a remarkable specificity profile that implements strong attenuation in nonmelanoma cells, including normal fibroblasts and keratinocytes.

Transcriptional targeting of adenoviral vector through the CXCR4 tumor-specific promoter.

Adenoviral vectors are considered to be good gene delivery vectors for cancer gene therapy due to their wide host tissue range and cell cycle-independent infectivity. However, the disadvantages include the lack of specificity for cancer cells and the high liver accumulation in vivo. The human CXCR4 gene is expressed at high levels in many types of cancers, but is repressed in the liver. We explored the CXCR4 promoter as a candidate to restrict adenoviral transgene expression to tumor cells with a low expression in host tissues. The luciferase activities in multiple cancer cell lines infected with recombinant adenovirus reAdGL3BCXCR4 or the control vector reAdGL3BCMV revealed that the CXCR4 promoter exhibited relatively high transcriptional activity in a breast cancer cell line, MDA-MB-361, and two ovarian cancer cell lines, OVCAR-3 and SKOV3. ip1, 65% (P=0.0087), 16.7% (P=0.1) and 20% (P=0.0079) compared to that of the CMV promoter, respectively, and low expression, 4.9 and 0.1%, respectively, in both normal cell lines HFBC and HMEC. In addition, CXCR4 had a low expression of luciferase (0.32%) compared to that of the CMV promoter in mouse liver in vivo. The data also revealed that the CXCR4 promoter was a stronger tumor-specific promoter (TSP) than the Cox-2M promoter in primary melanomas obtained from two patients. The CXCR4 promoter is shown to have a 'tumor-on' and 'liver-off' status in vitro and in vivo, and CXCR4 may prove to be a good candidate TSP for cancer gene therapy approaches for melanoma and breast cancers.

Combined transcriptional and transductional targeting improves the specificity and efficacy of adenoviral gene delivery to ovarian carcinoma.

Adenoviruses are efficient gene delivery vehicles but have broad native tropism. To this end, finding ways to target this virus specifically to carcinomas has become an important focus of cancer gene therapy. Transductional and transcriptional forms of targeting have been used with promising results in ovarian carcinoma. Therefore, we combined both forms of targeting to investigate the effect on the specificity and efficiency of transgene expression in this disease. We used the tissue-specific SLPI promoter and the ovarian cancer associated targeting adaptor protein, sCARfC6.5. This bispecific protein contains the coxsackie-adenovirus receptor ectodomain and a single-chain antibody specific for c-erbB-2. Viruses containing the SLPI or the ubiquitously expressed CMV promoter, with or without sCARfC6.5, were used for infection of ovarian cancer cell lines, primary ovarian tumor cells, and in an orthotopic model of disseminated ovarian carcinoma. This dual-targeting strategy increased the efficiency and specificity of transgene expression in vitro in reporter and cell-killing assays, and in vivo. By using both the SLPI promoter and sCARfC6.5, transgene expression was increased in ovarian tumors and decreased in normal tissues, including the liver. Thus, we show that combining transcriptional and transductional targeting can increase the efficacy and specificity of adenoviral gene therapy for ovarian carcinoma.

Cyclooxygenase-2 promoter for tumour-specific targeting of adenoviral vectors to melanoma.

Novel therapeutic strategies are warranted for the treatment of metastatic melanoma as conventional therapies are inefficient. Conceptually, these strategies should be systemic and tumour-targeted. Gene therapy and viral oncolysis represent promising new approaches for cancer treatment that allow for the incorporation of molecular targeting strategies. In this regard, we analysed cyclooxygenase-2 (cox-2) expression as a potential new target for melanoma gene therapy. By reverse transcription polymerase chain reaction analysis, we showed cox-2 mRNA expression in all of the six tested melanoma cell lines, thus establishing cox-2 as a tumour marker for melanoma of potential interest for targeted therapeutics. Next, we analysed the activity and specificity of the cox-2 promoter within adenoviral vectors by luciferase assays. For this purpose, melanoma cell lines, primary melanoma cells and normal melanocytes were infected with adenoviruses containing cox-2 promoter sequences driving the luciferase reporter gene. The results demonstrated activity of the cox-2 promoter in melanoma cell lines and primary melanoma cells, but not in non-malignant primary epidermal melanocytes. Thus, we established herein the tumour specificity of the cox-2 promoter with potential applications for transcriptional targeting of adenoviral vector-based cancer gene therapy or virotherapy to melanoma.

Metabolism of the styrene metabolite 4-vinylphenol by rat and mouse liver and lung.

Styrene is a widely used chemical in the reinforced plastics industry and in polystyrene production. Its primary metabolic pathway to styrene oxide and then to styrene glycol, which is further metabolized to mandelic acid and phenylglyoxylic acid, has been well studied. However, a few studies have reported finding a minor metabolite, 4-vinylphenol (4-VP), in rat and human urine. The present studies sought to determine if the formation and metabolism of 4-VP in rat and mouse liver and lung preparations could be measured. When styrene was incubated with hepatic and pulmonary microsomal preparations, 4-VP formation could not be measured in these preparations. However, considerable 4-VP metabolizing activity, as determined by the loss of 4-VP, was observed in both mouse and rat liver and lung microsomal preparations. 4-Vinylphenol metabolizing activity in mouse liver microsomes was three times greater than that in rat liver microsomes, and activity in mouse lung microsomes was eight times greater than that in rat lung microsomes. This activity was completely absent in the absence of NADPH. Studies with cytochrome P-450 inhibitors indicated the involvement of CYP2E1 and CYP2F2. Induction of CYP2E1 by pyridine resulted in an increase in 4-VP metabolism by mouse hepatic microsomes but not by pulmonary microsomes. The metabolite(s) formed as a result of this oxidative pathway remain to be identified. In additional studies, glutathione conjugation appeared to be involved in 4-VP metabolism with the highest activity being in mouse lung, with or without the addition of NADPH.

Hyperglycemia alters cytoplasmic Ca2+ responses to capacitative Ca2+ influx in rat aortic smooth muscle cells.

Concentrations of free cytoplasmic Ca2+ in rat aortic smooth muscle (RASM) cells were monitored using the ratiometric Ca2+ indicator fura 2-acetoxymethyl ester (AM). In RASM cells cultured in 5 mM Glc, incubation with angiotensin II, ATP, or thapsigargin [a selective inhibitor of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase] depleted SR Ca2+ stores and initiated a capacitative Ca2+ influx through the plasma membrane. This influx was resistant to verapamil, a selective inhibitor of L-type voltage-gated Ca2+ channels, but was sensitive to SKF-96365, an inhibitor of the receptor-operated Ca2+ entry pathway. RASM cells cultured in 25 mM Glc exhibited a significant decrease in cytoplasmic Ca2+ responses to agonist-induced Ca2+ release from SR stores and to subsequent capacitative Ca2+ entry. In addition, the cytoplasmic response to thapsigargin-induced release of Ca2+ from the SR in hyperglycemic cells peaked more sharply than in control cells and returned to baseline more rapidly. The effects of hyperglycemia were not overcome by myo-inositol supplementation.

[Excessive population and health]. / La poblacion excesiva y la salud.

PIP: Population density in El Salvador is among the highest in the world. In metropolitan San Salvador and the other main cities, crowding, squatter settlements, unemployment and underemployment, scarcity of basic services, squalor, and other social pathologies appear to be increasing. Overpopulation poses an enormous challenge for development. Reflection on the benefits of family planning has been delayed in El Salvador, and in the interim there have been increases in social inequality, misery, and hunger. Family planning programs have been referred to as "neo-Malthusian" and contrary to the right to life, but in fact they promote birth spacing and free selection of methods by couples, contributing to improvement in the quality of family life. Family planning allows couples to limit their offspring to those they can adequately care for emotionally and materially. People must be shown that family planning alleviates many of humanity's problems.^ieng

[Deforestation and overpopulation]. / Desforestacion y sobrepoblacion.

PIP: Ecological damage and irrational deforestation in El Salvador are very serious problems with potentially catastrophic consequences in the near future. Each year the amount of rainfall declines precipitously, the rivers lose volume, temperatures rise to levels unheard of in the rainy season, and ecological balance is disturbed, with disappearance of entire species of plants and animals. Desertification threatens future generations of Salvadorans. The causes of deforestation are multiple, but ill-conceived urbanization policy and immoderate population growth are two important factors. The increased population requires new housing, electric lines, aqueducts, clinics, and schools. When conditions are unfavorable, marginal zones or squatter settlements lacking all these basic services proliferate. With their concentrations of waste and environmental degradation, they are converted into foci of infectious disease and social pathology. Much scarce agricultural land has been lost to urban development as the population grows uncontrollably in the nation's small area.^ieng

Isolation and expression of a rat liver cDNA encoding phosphoglucomutase.

A cDNA encoding phosphoglucomutase (PGM) has been isolated from a rat liver cDNA library following screening with a polymerase chain reaction product. The cDNA was found to contain a 53-base-pair (bp) 5' untranslated region (5' UTR), a single start codon and consensus initiation sequence, an open reading frame (ORF) of 1686 bp, and a 3' untranslated tail. A comparison to the rabbit and human muscle PGM cDNAs [Whitehouse et al., Proc. Natl. Acad. Sci. USA 89 (1992) 411-415] showed 90% identity of rat cDNA to both, while a comparison to the deduced amino acid sequences showed 97 and 96% identity, respectively. Northern blot analyses determined that PGM was encoded by a single mRNA in rat liver, of approximately 2.2 kb. Following transfection of COS-7 cells with a plasmid containing the entire PGM ORF, indirect immunofluorescence analyses using a PGM-specific monoclonal antibody determined that approximately 5% of the cells displayed 50-100 times greater fluorescence than that seen in the remainder of the cells or in mock transfects. The enhanced production of PGM was also demonstrated by Western blotting and by enzymatic activity assays.

beta-Phosphorothioate analogs of nucleotide sugars are resistant to hydrolytic degradation and utilized efficiently by glycosyltransferases.

The alpha- and beta-phosphorothioate analogs of UDP-Gal and UDP-Glc, in which a sulfur is exchanged for a non-bridging oxygen at one of the phosphate groups, have been synthesized and tested for their resistance to enzymatic degradation and for their usefulness in glycosyltransferase reactions. The alpha analogs were found to be no more resistant to hydrolysis than the native nucleotide sugars, but as previously reported (R. B. Marchase et al. (1987) Biochim. Biophys. Acta 916: 157) the beta S analogs were approximately 10 times more resistant. The beta S analog and native UDP-Glc were found to have comparable Km's when used in assays for glucosylphosphoryl dolichol synthase with rat liver and hen oviduct microsomes, although the apparent Vmax of the reaction was about twofold higher for the analog, presumably due to its resistance to degradation. Partially purified 4 beta-galactosyltransferase exhibited a Vmax with (beta S)UDP-Gal that was only slightly lower than that with UDP-Gal and a Km that was slightly increased. The effectiveness of the analog was especially apparent in assays for 4 beta-galactosyltransferase on intact sperm and in rat liver homogenates, in which hydrolysis of the normal substrate was very rapid and net incorporation was at least 4 times greater with the beta S analog in each system.

Cell surface expression of 4 beta-galactosyltransferase accompanies rat parotid gland acinar cell transition to growth.

Rat parotid gland acinar cells stimulated to divide by a chronic regimen of isoproterenol demonstrate a dramatic increase in the synthesis of the glycosyltransferase 4 beta-galactosyltransferase. A plasma membrane localization for much of the increase in 4 beta-galactosyltransferase was determined by density gradient membrane fractionation. Golgi-enriched fractions showed no increase in specific activity, while plasma membrane activity increased 40-fold. This selective increase at the cell surface was confirmed by immunofluorescence of intact, nonpermeabilized cells from treated glands, using a monospecific antibody prepared against the purified bovine milk transferase. In detergent-permeabilized cells staining of nontreated cells was seen only as groups of perinuclear vesicles, presumed to be Golgi apparatus. In isoproterenol-treated and permeabilized cells both presumptive Golgi and cell surface staining was apparent. Enzyme assays performed on intact cells established that the enzyme's active site was oriented to the exterior of the cells. The transferase could be detected as early as 3 hr after the primary challenge with isoproterenol. Pretreatment of rats with cycloheximide prevented its appearance.

The beta-phosphoro[35S]thioate analogue of UDP-Glc is efficiently utilized by the glucose phosphotransferase and is relatively resistant to hydrolytic degradation.

The beta-phosphoro[35S]thioate analogue of UDP-glucose ((beta-35S)UDP-Glc) is utilized with approximately the same efficiency as the parent compound by the UDP-glucose:glycoprotein glucose-1-phosphotransferase (glucosyltransferase), which catalyzes the transfer of alpha Glc-1-P from UDP-Glc to mannose-containing oligosaccharides on acceptor glycoproteins. The same endogenous acceptor glycoproteins are labeled by the glucosyltransferase using [beta-32P]UDP-Glc and (beta-35S)UDP-Glc. However, in liver homogenates, incorporation from [beta-32P]UDP-Glc ceases to increase after about 4 min of incubation, while incorporation from (beta-35S)UDP-Glc persists for at least 1 h. This difference is due to an approx. 10-fold slower hydrolytic rate for the phosphorothioate analogue than for the parent compound, a finding similar to previous work showing that a variety of nucleases and phosphodiesterases are less efficient in cleaving phosphorothioate DNA than the native polymer.

Malathion for treatment of Pediculus humanus var capitis infestation.

One hundred fifteen subjects took part in a double-blind study comparing the efficacy and safety of 0.5% malathion lotion (Prioderm lotion) and the alcoholic vehicle of the lotion as pediculicides in the treatment of head lice. The subjects, with a mean age of 9 years, had long-standing lice infestations and at least 20 viable ova. The subjects were treated with the test preparation, which was allowed to remain on the hair for 12 hours. Ovicidal and pediculicidal efficacy for the malathion lotion was significant; overall clinical evaluation by the investigators showed a significant number of moderate to marked therapeutic responses to malathion lotion. Only one adverse reaction (in the malathion lotion group) was reported, the stinging of pyodermal lesions in reaction to the alcoholic vehicle. Other concomitant scalp diseases were unchanged or improved at the end of the study.