Protease pretreatment increases the efficacy of adenovirus-mediated gene therapy for the treatment of an experimental glioblastoma model.

Effective virus-mediated gene therapy for cancer will be facilitated by procedures that enhance the low level of gene transfer mediated by replication-deficient, recombinant viral vectors. We found recently that protease pretreatment of solid tumors is a useful strategy for enhancing virus-mediated gene transduction in vivo. In this study, we examined the potential of protease pretreatment to improve the efficacy of a gene therapy strategy for prodrug activation that depends on infection with a recombinant adenovirus encoding herpes simplex virus thymidine kinase (Ad-HSV-tk). Trypsin or a dissolved mixture of collagenase/dispase was inoculated into xenografts derived from the human glioblastoma multiforme-derived cell lines, U87 or U251. Ad-HSV-tk was administered 24 h after protease pretreatment, and animals were then treated for 10 days with ganciclovir (GCV). We found that protease pretreatment increased the efficacy of adenovirus mediated HSV-tk/GCV gene therapy in these experimental tumor models. Mice receiving Ad-HSV-tk/GCV after protease pretreatment demonstrated a significantly greater regression of tumors compared with those treated with Ad-HSV-tk/GCV alone. No adverse effects of protease pretreatment were observed. No signs of metastasis were seen either by histological inspection of lymph nodes or by a PCR-based analysis of selected mouse tissues to detect human tumor cells. Our findings indicate that protease pretreatment may be a useful strategy to enhance the efficacy of virus-mediated cancer gene therapy.

Id gene expression as a key mediator of tumor cell biology.

Id genes encode members of the helix-loop-helix (HLH) family of transcription factors that inhibit transcription by forming inactive heterodimers with basic HLH (bHLH) proteins. There are four members of the Id gene family recognized in mammals, and the proteins they encode share homology primarily in their HLH domain. bHLH proteins typically form heterodimers with other bHLH proteins, and their basic domain binds to a DNA sequence element, the E-box, activating transcription. Products of Id genes lack the basic DNA binding domain of the bHLH transcription factors, and when they heterodimerize with bHLH proteins, the complexes are inactive. Generally, high levels of Id mRNA are detected in proliferative undifferentiated, embryonal cells and lower levels are detected in well-differentiated, mature, adult tissues. In vitro, these genes are generally expressed at lower levels in cells after the induction of differentiation. Recently, high levels of expression of Id genes have been identified in cell lines derived from a wide variety of different tumors and in tumor tissues as well. These findings suggest that not only the inappropriate proliferation of tumors but also the anaplastic characteristics that contribute to their malignant behavior may be regulated by Id gene expression.

Pretreatment with protease is a useful experimental strategy for enhancing adenovirus-mediated cancer gene therapy.

A key impediment to the development of effective virus-mediated gene therapy for cancer is the low level of gene transfer that occurs after the administration of recombinant viral vectors. Improving in vivo infection and transduction efficiency is an important goal for gene therapy. The limited distribution of gene delivery is particularly problematic when large vectors such as recombinant adenoviruses and retroviruses are used to mediate transgene delivery to solid tumors. To facilitate the spread of virus, we have investigated the potential of administering proteases prior to the intratumoral inoculation of recombinant replication deficient adenovirus. For these studies, we chose proteases that are active against collagen and the other extracellular matrix proteins found in primary brain tumor tissue, but are not widely expressed in normal brain. Various concentrations of a mixture of collagenase/dispase or trypsin were inoculated into xenografts of human glioblastoma multiforme-derived brain tumor cell lines U87, U251, and SF767. Subsequently, recombinant adenovirus encoding the beta-galactosidase gene was administered and tumor tissue was examined for evidence of virus infection. Both collagenase/dispase and trypsin enhanced virus infection, indicating that protease pretreatment may be a useful strategy for enhancing virus-mediated gene transduction for many in vivo applications.

Overexpression of E2F1 in glioma-derived cell lines induces a p53-independent apoptosis that is further enhanced by ionizing radiation.

Glioma cell lines show variable responses to radiation in a manner influenced by their p53 status. Irradiation of glioma cell lines does not generally induce apoptosis. When wild-type p53 is present, these cells undergo a G1 arrest that is closely associated with increased radiosensitivity as measured by clonogenic survival. Previously, others have shown that dysregulated overexpression of E2F1 induces apoptosis in cell lines with either functional or inactivated p53. We found that regardless of p53 status, apoptosis induced by overexpression of E2F1 in glioma cell lines was further enhanced by treatment with ionizing radiation. BAX induction did not follow E2F1 overexpression or irradiation in the glioma cell lines tested. Thus, the apoptotic response of glioma-derived cells to irradiation can be enhanced by E2F1 by a mechanism that does not involve the induction of BAX.

Effects of age and exercise training on size and composition of the rat left main coronary artery.

This study evaluated the effects of age and exercise training on the left main coronary artery (LMCA) in young (Y-5 months) and old (O-27.5 months) female Fischer 344 rats. Both age groups were divided into trained (T) and weight-matched sedentary (S) control groups. Training consisted of 10 weeks of treadmill running progressing to a maximum workload of 15% grade, 1 hr/day, 5 d/wk at speeds of 36 and 15 m/min for the Y and O rats, respectively. Aging resulted in a 40% increase in left ventricle (LV) weight which was proportional to the increased body weight of the old animals. Exercise training produced a mild (approximately 10%) but significant left ventricular hypertrophy (LVH) in both trained groups. Cross-sectional area of the LMCA lumen and wall, wall thickness, and areas of collagen (C), elastin (E), and collagen-to-elastin ratio (C/E) of the LMCA wall were determined morphometrically in all four groups. A method for pinpointing the coronary ostium for use as a reference point was also developed. LMCA lumen area almost doubled (p < .001) across the measured age difference, but was unaffected by training. With aging, the increase in LMCA wall area bordered on significance (p < .053), while wall thickness, C area, and the C/E ratio were unchanged. Our results indicate that there is a disproportionate increase in the cross-sectional area of the rat LMCA with respect to LV mass changes with aging. This finding presumably reflects adaptation of this vessel to elevated resistances further downstream in the coronary circulation so that tissue perfusion can be maintained.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II stimulates ammoniagenesis in canine renal proximal tubule segments.

To determine whether angiotensin II (ANG II) affects ammoniagenesis in renal proximal tubule, ammonia production was measured in suspensions of canine renal proximal tubule segments (PCT) incubated with L-glutamine and varying concentrations of ANG II. Ammonia production from PCT was significantly increased by 15.5 +/- 1.1% in the presence of ANG II (10(-6) M) at 2 h. Similarly, glucose production significantly increased by 10.0 +/- 0.9%. Half-maximal stimulation occurred at approximately 10(-9) M ANG II. Stimulation of ammonia production by ANG II was blocked in the presence of the ANG II antagonist, [Sar1-Ile8]ANG II (10(-6) M). Enhancement of ammonia production in PCT by ANG II occurred in acidotic and neutral media but not in alkalotic medium. When extracellular [Na+] = intracellular [Na+] ANG II significantly increased ammonia production in PCT. Absence of extracellular Ca2+ or addition of trifluoperazine or N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) (Ca2(+)-calmodulin-dependent pathway inhibitors) blocked the action of ANG II to enhance ammonia production. We conclude that ANG II stimulates ammonia and glucose production in canine renal PCT via a receptor-mediated signal. The action of ANG II on ammoniagenesis may be mediated by a calcium-calmodulin-dependent pathway. Stimulation of ammoniagenesis in vitro under normal and acidotic conditions may reflect a role in vivo for ANG II in the regulation of renal acid-base metabolism.

Growth hormone regulates ammoniagenesis in canine renal proximal tubule segments.

To determine whether growth hormone (GH) directly affects ammoniagenesis in the renal proximal tubule, ammonia production was measured in suspensions of isolated canine renal proximal tubule segments (IPTs) incubated with 2.5 mM L-glutamine and varying concentrations of human growth hormone (hGH). Ammonia production from IPTs significantly increased by nearly threefold in the presence of hGH (10(-6) M) at 60 min. This increase was dose dependent, with as little as 10(-9) M hGH significantly stimulating ammonia production. In addition, hGH enhanced glucose production when lactate, alanine, and succinate replaced L-glutamine as substrate. hGH significantly stimulated ammonia production when IPTs were incubated at alkalotic and neutral pH. The effect of hGH was lost at acidic pH. When hGH was added to IPTs incubated under Na(+)-equilibrated conditions, ammonia production was not different from control. hGH stimulated ouabain-sensitive Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity by 8.1 +/- 1.1% in basolateral membranes isolated from IPTs. hGH stimulation of proximal tubule ammonia production from L-glutamine occurs at physiological concentrations of hGH and when the extracellular-to-intracellular Na+ gradient favors L-glutamine transport. This effect is associated with an increase in basolateral Na(+)-K(+)-ATPase activity. The data suggest a role for hGH in the regulation of renal acid-base metabolism under physiological conditions in which increased net acid excretion is important.

Activated neutrophils inhibit Na(+)-K(+)-ATPase in canine renal basolateral membrane.

To examine the effects of activated neutrophils (PMNs) on Na(+)-K(+)-ATPase, phorbol 12-myristate 13-acetate (PMA)-stimulated PMNs were incubated with canine renal cortical basolateral membrane (BLM), and BLM ouabain-sensitive Na(+)-K(+)-ATPase activity was subsequently quantified. Na(+)-K(+)-ATPase activity decreased to 40.0 +/- 8.7% (SE) of control in the presence of activated PMNs, from 0.89 +/- 0.12 to 0.34 +/- 0.05 mumol Pi.mg protein-1.min-1. This inhibition coincided with a decrease in the apparent Michaelis constant (Km) for ATP from 0.18 +/- 0.02 to 0.05 +/- 0.01 mM. Inclusion of catalase (CAT) and superoxide dismutase (SOD) in the BLM/PMN/PMA incubation mixture resulted in partial preservation of enzyme activity, with an increase to 57.0 +/- 4.6% of control with CAT alone and to 70.0 +/- 5.3% with both CAT and SOD. SOD alone had no protective effect. Neither the myeloperoxidase inhibitor azide nor the hypochlorous acid scavenger L-methionine preserved enzyme activity. Hydroxyl radical scavengers and iron chelators were also ineffective in attenuating Na(+)-K(+)-ATPase inhibition by activated PMNs. These results indicate that activated PMNs mediate a decrease in BLM Na(+)-K(+)-ATPase activity characterized by a reduction in maximum velocity and Km for ATP that appears to be mediated in part by reactive oxygen metabolites.

High affinity ligands from in vitro selection: complex targets.

Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an in vitro protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems.

The intrinsic radioresistance of glioblastoma-derived cell lines is associated with a failure of p53 to induce p21(BAX) expression.

Radiation is the primary modality of therapy for all commonly occurring malignant brain tumors, including medulloblastoma and glioblastoma. These two brain tumors, however, have a distinctly different response to radiation therapy. Medulloblastoma is very sensitive to radiation therapy, whereas glioblastoma is highly resistant, and the long-term survival of medulloblastoma patients exceeds 50%, while there are few long-term survivors among glioblastoma patients. p53-mediated apoptosis is thought to be an important mechanism mediating the cytotoxic response of tumors to radiotherapy. In this study, we compared the response to radiation of five cell lines that have wild-type p53: three derived from glioblastoma and two derived from medulloblastoma. We found that the medulloblastoma-derived cell lines underwent extensive radiation-induced apoptotic cell death, while those from glioblastomas did not exhibit significant radiation-induced apoptosis. p53-mediated induction of p21(BAX) is thought to be a key component of the pathway mediating apoptosis after the exposure of cells to cytotoxins, and the expression of mRNA encoding p21(BAX) was correlated with these cell lines undergoing radiation-induced apoptosis. The failure of p53 to induce p21(BAX) expression in glioblastoma-derived cell lines is likely to be of biologic significance, since inhibition of p21(BAX) induction in medulloblastoma resulted in a loss of radiation-induced apoptosis, while forced expression of p21(BAX) in glioblastoma was sufficient to induce apoptosis. The failure of p53 to induce p21(BAX) in glioblastoma-derived cell lines suggests a distinct mechanism of radioresistance and may represent a critical factor in determining therapeutic responsiveness to radiation in glioblastomas.

Enrichment for RNA molecules that bind a Diels-Alder transition state analog.

RNA molecules that bind a transition state analog for a Diels-Alder reaction (Kd = 0.35 +/- 0.05 mM) were isolated from a starting pool of approximately 10(14) sequences by affinity chromatography. After the initial rise and plateau of the amount of RNA that eluted with soluble analog, a step gradient elution was used to further enrich the pool for sequences with higher affinities for the target. To our knowledge, the isolation of RNA molecules that bind either a nonplanar or a hydrophobic ligand has not been reported previously. A conserved nucleotide sequence and secondary structure present in many of the RNA molecules are necessary but not sufficient for binding the analog. No catalysts of the targeted Diels-Alder reaction were found among the binders. The absence of catalysis contrasts with previous successful experiments with antibodies and suggests that other strategies may be needed to identify oligonucleotides with diverse catalytic activities.