Sustained p16INK4a expression is required to prevent IR-induced tumorigenesis in mice.

Exposure of murine and human tissues to ionizing radiation (IR) induces the expression of p16INK4a, a tumor suppressor gene and senescence/aging biomarker. Increased p16INK4a expression is often delayed several weeks post exposure to IR. In this context, it remains unclear if it occurs to suppress aberrant cellular growth of potentially transformed cells or is simply a result of IR-induced loss of tissue homeostasis. To address this question, we used a conditional p16INK4a null mouse model and determined the impact of p16INK4a inactivation long-term post exposure to IR. We found that, in vitro, bone marrow stromal cells exposed to IR enter DNA replication following p16INK4a inactivation. However, these cells did not resume growth; instead, they mostly underwent cell cycle arrest in G2. Similarly, delayed inactivation of p16INK4a in mice several weeks post exposure to IR resulted in increased BrdU incorporation and cancer incidence. In fact, we found that the onset of tumorigenesis was similar whether p16INK4a was inactivated before or after exposure to IR. Overall, our results suggest that IR-induced p16INK4a dependent growth arrest is reversible in mice and that sustained p16INK4a expression is necessary to protect against tumorigenesis.

Exaggerated vasomotor response to ANG II in rats with fetal programming of hypertension associated with exposure to a low-protein diet during gestation.

The renin-angiotensin system plays a key role in the initiation and maintenance of elevated blood pressure associated with altered intrauterine milieu. The current studies were undertaken to verify whether vascular response to ANG II is increased in adult offspring of low-protein fed dams (LP) compared with control (CTRL) and if so, to examine underlying mechanism(s). ANG II-induced contraction of carotid rings was increased in LP (E(max), the maximum asymptote of the curve, relative to maximal response to KCl 80 mM: 230 +/- 3% LP vs. 201 +/- 2% CTRL, P < 0.05). In both groups, contraction to ANG II was mediated solely by AT1R. Responses to thromboxane A2 analog U-46619 and to KCl 80 mM under step increases in tension were similar between groups. Endothelium depletion enhanced contraction to ANG II in both groups, more so in LP. Blockade of endothelin formation had no effect on response to ANG II, and ANG-(1-7) did not elicit vasomotor response in either group. Superoxide dismutase (SOD) analog Tempol normalized LP without modifying CTRL response to ANG II. Basal levels of superoxide (aortic segments, lucigenin-enhanced chemiluminescence and fluorescent dye hydroethidine) were higher in LP. ANG II further increased superoxide production in LP only, and this was inhibited by coincubation with diphenylene iodonium or apocynin (inhibitor of NADPH oxidase complex). AT1R expression in carotid arteries was increased in LP, whereas SOD expression was unchanged. In conclusion, vasoconstriction to ANG II is exaggerated in this model of developmental programming of hypertension, secondary to enhanced vascular production of superoxide anion by NADPH oxidase with concomitant increase of AT1R expression.

Microvascular rarefaction and decreased angiogenesis in rats with fetal programming of hypertension associated with exposure to a low-protein diet in utero.

In hypertension, increased peripheral vascular resistance results from vascular dysfunction with or without structural changes (vessel wall remodeling and/or microvascular rarefaction). Humans with lower birth weight exhibit evidence of vascular dysfunction. The current studies were undertaken to investigate whether in utero programming of hypertension is associated with in vivo altered response and/or abnormal vascular structure. Offspring of Wistar dams fed a normal (CTRL) or low (LP)-protein diet during gestation were studied. Mean arterial blood pressure response to ANG II was significantly increased, and depressor response to sodium nitroprusside (SNP) infusions significantly decreased in male LP adult offspring relative to CTRL. No arterial remodeling was observed in male LP compared with CTRL offspring. Capillary and arteriolar density was significantly decreased in striated muscles from LP offspring at 7 and 28 days of life but was not different in late fetal life [day 21 of gestation (E21)]. Angiogenic potential of aortic rings from LP newborn (day of birth, P0) was significantly decreased. Striated muscle expressions (Western blots) of ANG II AT(1) receptor subtype, endothelial nitric oxide synthase, angiopoietin 1 and 2, Tie 2 receptor, vascular endothelial growth factor and receptor, and platelet-derived growth factor C at E21 and P7 were unaltered by antenatal diet exposure. In conclusion, blood pressure responses to ANG II and SNP are altered, and microvascular structural changes prevail in this model of fetal programming of hypertension. The capillary rarefaction is absent in the fetus and appears in the neonatal period, in association with decreased angiogenic potential. The study suggests that intrauterine protein restriction increases susceptibility to postnatal factors resulting in microvascular rarefaction, which could represent a primary event in the genesis of hypertension.

Selection of drug-resistant transduced cells with cytosine nucleoside analogs using the human cytidine deaminase gene.

Hematopoietic toxicity produced by most anticancer drugs limits their potential for curative therapy. We have shown previously that the human cytidine deaminase (CD) gene can confer drug resistance in murine bone marrow cells (BMCs) to the nucleoside analog, cytosine arabinoside (ARA-C). In the present study, as the first objective we showed that the CD gene can also render drug resistance in BMCs to related analogs, 2',2'-difluorodeoxycytidine (dFdC) and 5-azadeoxycytidine (5-AZA-CdR). As a second objective, we investigated the potential of ex vivo selection with cytosine nucleoside analogs of CD-transduced BMC. The goal of this approach was to enrich the fraction of CD-transduced BMCs so as to increase the transgene expression and level of drug resistance before transplantation. This strategy may have the potential to circumvent the problem in clinical gene therapy of low level of gene transfer and adequate long-term gene expression. Using a bicistronic retroviral vector containing the CD and the green fluorescent protein (CDiGFP), we transduced murine L1210 leukemic cells. All three analogs, ARA-C, dFdC, and 5-AZA-CdR were demonstrated in vitro to enrich (>95%) the population of leukemic cells expressing the GFP transgene. However, with CD-transduced primary murine BMCs cultivated at high cell density we observed that in vitro selection with ARA-C was not possible due to release of CD into the culture medium at amounts that were sufficient to inactivate the analog. The CD-containing medium produced a chemoprotective effect on mock BMCs as shown by lack of significant growth inhibition in the presence of ARA-C. However, at low cell density in a cell mixture containing CD-transduced cells, the mock BMCs showed marked drug sensitivity to ARA-C as determined by clonogenic assay. Selection with ARA-C was shown to significantly increase the CD enzyme activity in transduced BMC. These results suggest that CD gene has the potential to be a good selectable marker and a possible tool for chemoprotection in cancer gene therapy.

Elimination of 7-aminoflunitrazepam and flunitrazepam in urine after a single dose of Rohypnol.

The hypnotic benzodiazepine flunitrazepam (Rohypnol) has been identified as the drug of choice for the purposes of "drugging" unsuspecting victims and raping them while they are under the influence of this substance. The objective of this paper was to study elimination of flunitrazepam and 7-aminoflunitrazepam in urine collected from ten healthy volunteers who received a single 2 mg oral dose of Rohypnol, to determine how long after drug administration 7-aminoflunitrazepam can be detected. A highly sensitive NCI-GC-MS method for the simultaneous quantitation of flunitrazepam (LOQ 100 pg/mL) and 7-aminoflunitrazepam (LOQ 10 pg/mL) in urine was developed. All samples were screened for benzodiazepines using optimized micro-plate enzyme immunoassay. The highest concentrations of 7-aminoflunitrazepam (70-518 ng/mL) and flunitrazepam (0.7-2.8 ng/mL) in urine were observed 6 h after drug administration in nine subjects and after 24 h in one subject. In six subjects 7-aminoflunitrazepam was detected up to 14 days after flunitrazepam administration, in one subject up to 21 days and in three subjects up to 28 days. In urine samples collected from six volunteers, flunitrazepam was detected three days after Rohypnol intake, in three subjects 24 h, and in one subject 5 days later. Benzodiazepine micro-plate enzyme immunoassay kit allowed the detection of flunitrazepam and metabolities 5 to 21 days after drug administration.

DNA methylation of retinoic acid receptor beta in breast cancer and possible therapeutic role of 5-aza-2'-deoxycytidine.

The retinoic acid receptor beta (RARbeta), a putative tumor suppressor gene, has been reported to be poorly expressed in breast cancer. In this report using the methylation-specific PCR reaction we observed DNA methylation in the promoter region of RARbeta in several primary breast tumors. DNA sequence analysis showed that the positions of 5-methylcytosine in the RARbeta promoter region was almost identical to that reported previously by our laboratory for human DLD-1 colon carcinoma cells (Anti-Cancer Drugs 1998; 9: 743). Several other cancer-related genes have been also reported to be silenced by DNA methylation, including the p16 tumor suppressor gene, E-cadherin, an invasion suppressor gene and the estrogen receptor gene in breast cancer cell lines. Since breast cancer cells have several potential target genes for the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-Aza-CdR), we investigated the in vitro antineoplastic activity of this analog on the human breast cancer cell line MDA-MB-231. We report that 5-Aza-CdR is a potent growth inhibitor and a potent cytotoxic agent against the breast carcinoma cells. These results suggest that 5-Aza-CdR may be an interesting agent to investigate in patients with breast cancer resistant to conventional chemotherapy.

Coexpression of cytidine deaminase and mutant dihydrofolate reductase by a bicistronic retroviral vector confers resistance to cytosine arabinoside and methotrexate.

The transfer of a drug resistance gene into hematopoietic cells is an approach being investigated to overcome the problem of myelosuppression produced by anticancer drugs. Chemotherapeutic agents are often given in combination in order to increase their effectiveness. Consequently, there is an advantage in designing vectors for gene transfer that are capable of expressing two drug resistance genes. We have constructed a bicistronic retroviral vector, MFG-DHFR-IRES/CD, which contains the mutated human dihydrofolate reductase (DHFR) cDNA with a phenylalanine-to-serine substitution at codon 31 (F31S) and the human cytidine deaminase (CD) cDNA. Murine fibroblast and hematopoietic cells were transduced with this vector and evaluated for their resistance to methotrexate (MTX) and cytosine arabinoside (ARA-C). The transduced fibroblast cells showed high levels of resistance to MTX and to ARA-C as determined by a clonogenic assay. Using enzymatic assays, we observed a coordinate increase in resistance to MTX and DHFR enzyme activity following an ARA-C selection. In addition, MTX selection produced an increase in CD enzyme activity and ARA-C resistance. Murine hematopoietic cells transduced with the bicistronic vector also showed drug resistance to both MTX and ARA-C. Interestingly, the double-gene construct conferred an equivalent level of drug resistance compared with single-gene vectors bearing only CD or DHFR genes in the hematopoietic cells. These results demonstrate the potential of the MFG-DHFR-IRES/CD vector to confer drug resistance to both MTX and ARA-C and may have future application in chemoprotection of normal hematopoietic cells in patients with cancer.

Retroviral transfer and long-term expression of human cytidine deaminase cDNA in hematopoietic cells following transplantation in mice.

The chemotherapeutic effectiveness of cytosine nucleoside analogues used in cancer therapy is limited by their dose-dependent myelosuppression. A way to overcome this problem would be to insert the drug-resistance gene, cytidine deaminase (CD), into normal hematopoietic cells. CD catalyzes the deamination and pharmacological inactivation of cytosine nucleoside analogues, such as cytosine arabinoside (Ara-C). The objective of this study was to determine if we could obtain long-term persistence and expression of proviral CD in hematopoietic cells following transplantation of CD-transduced bone marrow cells in mice. Murine hematopoietic cells were transduced with an MFG retroviral vector containing CD cDNA and transplanted into lethally irradiated mice. The recipient mice were administered three courses of 10-15 h i.v. infusions of Ara-C (75-110 mg/kg). Blood, marrow and spleen samples were obtained and analyzed for CD proviral DNA by PCR, CD activity by enzyme assay, and drug resistance to Ara-C by clonogenic assay. We detected the presence of the CD proviral DNA in most of the samples examined. Approximately 1 year after transplantation several mice showed increased expression of CD activity in these tissues and some mice displayed signs of Ara-C resistance. These data demonstrate that persistent in vivo expression of proviral CD can be achieved in transduced hematopoietic cells and indicate some potential of this gene for chemoprotection to improve the efficacy of cytosine nucleoside analogues in cancer therapy.

Neurochemical and electrophysiological studies on the inhibitory effect of ammonium ions on synaptic transmission in slices of rat hippocampus: evidence for a postsynaptic action.

To elucidate the mechanisms involved in the inhibition of synaptic transmission by ammonium ions, the effects of NH4Cl on glutamate release and on synaptic transmission from Schaffer collaterals to CA1 pyramidal cells were measured in fully submerged slices of rat hippocampus. The large, Ca(2+)-dependent release of glutamate evoked by electrical-field stimulation or by 56 mM K+ was not reduced by 5 mM NH4Cl. In contrast, 5 mM NH4Cl decreased the smaller, field stimulation-induced release of glutamate observed in the presence of low concentrations of Ca2+ (0.1 mM), as well as the spontaneous release of glutamate both in normal and low Ca2+. Unlike the Ca(2+)-dependent release of glutamate, synaptic transmission was reversibly depressed even by 1 mM NH4 Cl. Firing of CA1 pyramidal cells evoked by iontophoretically applied glutamate was significantly inhibited by 2 or 5 mM NH4Cl. This depression was increased in the presence of 25 microM bicuculline. Results suggest that ammonium ions do not depress the Ca(2+)-dependent release of glutamate originating from synaptic vesicles, which is involved in synaptic transmission. Rather, ammonium ions inhibit synaptic transmission by a postsynaptic action, a conclusion strengthened by the inhibitory effect of NH4Cl on glutamate-induced firing. However, NH4Cl may inhibit the formation of cytoplasmic glutamate, the source of spontaneous and Ca(2+)-independent release.