Efficient induction of apoptosis by ONYX-015 adenovirus in human colon cancer cell lines regardless of p53 status.

The ONYX-015 virus is a mutated adenovirus that in theory selectively replicates and induces cytolysis in tumor cells lacking functional p53. The present study investigated whether ONYX-015 viral infection alone or in combination with conventional chemotherapeutic agents could significantly increase apoptosis in human colon cancer cell lines, regardless of p53 status, compared to untreated cells. A pair of colon cancer cell lines that differ only in their p53 status (RKO with wild-type p53 and RKOp53 with deficient p53) was tested. Two chemotherapeutic agents, 5-fluorouracil (5-FU) and CPT-11, were tested in combination with ONYX-015. Final concentrations of these agents corresponded to peak plasma levels achievable in patients. ONYX-015 concentration was 10 p.f.u./cell. In RKO and RKOp53 cell lines, ONYX-015 viral infection alone or in combination with 5-FU or CPT-11 induced a significant increase in apoptosis compared to chemotherapeutic agents alone, regardless of p53 status. Moreover, the combination of ONYX-015 and chemotherapeutics induced more apoptosis than chemotherapeutics alone in the two colon cancer cell lines independently of their p53 status. We conclude that ONYX-015 virus infection alone or in combination with 5-FU or CPT-11 induced apoptosis in human colon cancer cell lines, independently of p53 status.

Alterations in DNA repair and telomere maintenance mechanism affect response to porphyrins in yeast.

BACKGROUND: DNA quadruplex-interactive porphyrin TMPyP4, but not its isomer TMPyP2, inhibits telomerase activity and causes chromosome fusion in vivo, suggesting interference with telomere maintenance. MATERIALS AND METHODS: We examined effects of these porphyrins and hydroxyurea on growth rates of yeast Saccharomyces cerevisiae wild type and strains with defects in telomere maintenance and/or DNA repair pathways (mec1, tel1, rad9), telomere binding protein (cdc13), and anaphase control (pds1). RESULTS: Hydroxyurea (20 mM) decreased proliferation rates only in mec1 mutant and deletion strains. TMPyP4 (200 microM) decreased growth in all strains, especially in rad9delta and mec1delta. The growth inhibition by TMPyP4 showed low growth inhibition in strains defective in cdc13 and pds1. TMPyP2 sterically prevented from forming a planar species did not significantly inhibit growth of any strain. Overexpression of telomere binding protein Rap1 hypersensitized the mec1delta and tel1delta to TMPyP4. CONCLUSIONS: Telomere maintenance represents a viable target for anticancer agents.

Neuropeptide receptor status in human tumor cell lines.

Tumor types expressing a neuroendocrine phenotype secrete neuropeptides with paracrine or autocrine growth factor activity. The efficacy of these paracrine or autocrine loops depends on the expression of specific receptors on tumor cells. Once specific receptors are identified, specific neuropeptide antagonists disrupting paracrine and autocrine loops could be potential treatments in neuropeptide-secreting tumors. In the present study, 11 human tumor cell lines representing astrocytoma, lymphoma, and pancreatic, prostate, lung and colon carcinomas were examined for expression of five different neuropeptide receptors (cholecystokinin, neurotensin, vasopressin, tachykinine substance P and cannabinoid) using RT-PCR and radioligand binding. The presence of various neuropeptide receptors in different human cancer cell lines supports development of new antitumor treatments based on disruption of neuropeptide autocrine growth pathways.

5,6-Dihydro-5'-azacytidine (DHAC) affects estrogen sensitivity in estrogen-refractory human breast carcinoma cell lines.

BACKGROUND: There is little effective therapy for patients with hormone-refractory breast cancer. Hormone resistance is frequently due to the transcriptional inactivation of the estrogen receptor (ER) gene. We determined the effect of DHAC, a cytosine DNA methyltransferase (CMT) inhibitor, on the estrogen sensitivity in three human breast carcinoma cell lines with intermediate to low levels of estrogen receptor (ER) expression: MCF7 (adriamycin-sensitive), MCF7M/Adr (adriamycin-resistant), and MDA-435, and one ER+ cell line, ZR75-1. MATERIALS AND METHODS: Cells maintained in culture were exposed to DHAC or vehicle continuously for 14 days, then exposed to estradiol or tamoxifen and counted on day 21. RESULTS: Exposure to DHAC did not affect estrogen sensitivity in ZR-75-1 and MCF7M/Adr cells. DHAC treatment of MCF7 and MDA-435 cells resulted in significant (p < 0.05) growth stimulation in response to estrogen at 10(-6) M, and to growth modulation by tamoxifen at 10(-5) to 10(-7) M. CONCLUSIONS: These data suggest that DHAC can restore the estrogen sensitivity in ER-breast cancer. Thus, DHAC and other novel CMT inhibitors may have a clinical application in treating estrogen-refractory breast cancer patients by restoring the estrogen sensitivity and allowing these patients to respond again to conventional therapy with estrogen antagonists.

Effects of cationic porphyrins as G-quadruplex interactive agents in human tumor cells.

A series of cationic porphyrins has been identified as G-quadruplex interactive agents (QIAs) that stabilize telomeric G-quadruplex DNA and thereby inhibit human telomerase; 50% inhibition of telomerase activity was achieved in HeLa cell-free extract at porphyrin concentrations in the range < or = 50 microM. Cytotoxicity of the porphyrins in vitro was assessed in normal human cells (fibroblast and breast) and human tumor cells representing models selected for high telomerase activity and short telomeres (breast carcinoma, prostate, and lymphoma). In general, the cytotoxicity (EC50, effective concentration for 50% inhibition of cell proliferation) against normal and tumor cells was > 50 microM. The porphyrins were readily absorbed into tumor cell nuclei in culture. Inhibition of telomerase activity in MCF7 cells by subcytotoxic concentrations of TMPyP4 showed time and concentration dependence at 1-100 microM TMPyP4 over 15 days in culture (10 population doubling times). The inhibition of telomerase activity was paralleled by a cell growth arrest in G2-M. These results suggest that relevant biological effects of porphyrins can be achieved at concentrations that do not have general cytotoxic effects on cells. Moreover, the data support the concept that a rational, structure-based approach is possible to design novel telomere-interactive agents with application to a selective and specific anticancer therapy.

Telomere-interactive agents affect proliferation rates and induce chromosomal destabilization in sea urchin embryos.

Cationic porphyrins, which interact with guanine quadruplex (G4) telomeric folds, inhibit telomerase activity in human tumor cells. In this study, we have further examined effects of porphyrins and other telomere- and telomerase-interactive agents on proliferation rates and chromosome stability in a novel in vivo model, developing sea urchin embryos. We studied two porphyrins: (i) TMPyP4, a potent telomerase inhibitor; and (ii) TMPyP2, an isomer of TMPyP4 and an inefficient telomerase inhibitor, azidothymine (AZT), the reverse transcriptase inhibitor, antisense phosphorothioate oligonucleotide to telomerase RNA (TAG6) and a control scrambled sequence (ODN). TMPyP4, AZT and TAG6 (but not TMPyP2 or ODN) decreased the rates of cell proliferation and increased the percentage of cells trapped in mitosis. Nuclear localization of TAG6, but not of ODN, was demonstrated with 5'-fluoresceinated analogs of TAG6 and ODN. Formation of elongated chromosomes incapable of separating in anaphase, induced by TMPyP4, AZT and TAG6, closely resembled phenotypes resulting from telomerase template mutation or dominant negative TRF2 allele. Our data suggest that G4-interactive agents exert their antiproliferative effects via chromosomal destabilization and warrant their further development as valuable anticancer tools.

Pharmacokinetics of hydroxyurea in nude mice.

Extrachromosomal DNA is the predominant form of gene amplification in human tumors. Hydroxyurea (HU) concentrations of 100-150 microM have been promising in vitro for extrachromosomal DNA elimination. The study objective was to determine the HU dose-concentration relationship in nude mice with HU doses from 0 to 200 mg/kg. For HU t1/2 determination, mice were injected with HU 100 mg/kg. A plasma concentration of 159 microM was achieved and a t1/2 of 11.3 min determined. Based on these findings, In vivo elimination studies will require frequent administration of HU to maintain plasma concentrations from 100 to 150 microM.

Induction of differentiation in HL60 cells by the reduction of extrachromosomally amplified c-myc.

Oncogene amplification in tumor cells results in the overexpression of proteins that confer a growth advantage in vitro and in vivo. Amplified oncogenes can reside intrachromosomally, within homogeneously staining regions (HSRs), or extrachromosomally, within double minute chromosomes (DMs). Since previous studies have shown that low concentrations of hydroxyurea (HU) can eliminate DMs, we studied the use of HU as a gene-targeting agent in tumor cells containing extrachromosomally amplified oncogenes. In a neuroendocrine cell line (COLO 320), we have shown that HU can eliminate amplified copies of c-myc located on DMs, leading to a reduction in tumorigenicity in vitro and in vivo. To determine whether the observed reduction in tumorigenicity was due to differentiation, we next investigated whether HU could induce differentiation in HL60 cells containing extrachromosomally amplified c-myc. We compared the effects of HU, as well as two other known differentiating agents (dimethyl sulfoxide and retinoic acid), on c-myc gene copy number, c-myc expression, and differentiation in HL60 cells containing amplified c-myc genes either on DMs or HSRs. We discovered that HU and dimethyl sulfoxide reduced both c-myc gene copy number and expression and induced differentiation in cells containing c-myc amplified on DMs. These agents failed to have similar effects on HL60 cells with amplified c-myc in HSRs. By contrast, retinoic acid induced differentiation independent of the localization of amplified c-myc. These data illustrate the utility of targeting extrachromosomal DNA to modulate tumor phenotype and reveal that both HU and dimethyl sulfoxide induce differentiation in HL60 cells through DM elimination.

Elimination of extrachromosomally amplified MYC genes from human tumor cells reduces their tumorigenicity.

Oncogene amplification has been observed in a broad spectrum of human tumors and has been associated with a poor prognosis for patients with several different types of malignancies. Importantly, at biopsy, the amplified genes localize to acentric extrachromosomal elements such as double-minute chromosomes (DMs) in the vast majority of cases. We show here that treatment of several human tumor cell lines with low concentrations of hydroxyurea accelerates the loss of their extrachromosomally amplified oncogenes. The decreases in MYC copy number in a human tumor cell line correlated with a dramatic reduction in cloning efficiency in soft agar and tumorigenicity in nude mice. No effect on gene copy number or tumorigenicity was observed for a closely related cell line containing the same number of chromosomally amplified MYC genes. One step involved in the accelerated loss of extrachromosomal elements is shown to involve their preferential entrapment of DMs within micronuclei. The data suggest that agents that accelerate the loss of extrachromosomally amplified genes could provide valuable tools for moderating the growth of a large number of human neoplasms.

Effects of respiratory acidosis on HCO3- transport by rabbit collecting tubules.

These studies were performed to determine whether in vitro elevation of bath PCO2 with associated reduction in pH (acute respiratory acidosis) affected bicarbonate transport by isolated perfused rabbit cortical collecting tubules (CCT) and collecting tubules from the outer (OMCTos) and inner (OMCTis) stripes of the outer medulla. When the PCO2 was elevated and pH reduced from approximately 7.4 to 7.0 the rate of total CO2 absorption increased to 252% of that observed at pH 7.4 in CCT, 146% in OMCTos, and 150% in OMCTis. In OMCTis, pretreatment with colchicine inhibited the stimulation of total CO2 absorption associated with respiratory acidosis, whereas lumicolchicine did not. Similar inhibition was observed in the presence of maptam and a low calcium concentration and in the presence of a calmodulin inhibitor. No differences were observed in apical or basolateral membrane morphometry of principal or intercalated cells between control tubules and those subjected to respiratory acidosis. The results indicate that acute respiratory acidosis stimulates acidification by the rabbit distal nephron in vitro through a process(es) that, at least in OMCTis, evidently involves the cell cytoskeleton and changes in cell calcium and calmodulin activities.

Bicarbonate transport in collecting tubules from outer stripe of outer medulla of rabbit kidneys.

The purpose of this study is to characterize the features of bicarbonate (total CO2) transport in isolated perfused collecting tubules obtained from the outer stripe of the outer medulla (OMCTos) of rabbit kidneys. Under control conditions (25 mM HCO3- in the perfusate and bath), all OMCTos studied absorbed total CO2 at a mean rate of 8.61 +/- 0.44 pmol.mm-1.min-1. Ouabain (10(-4) M in the bath) did not affect the rate of total CO2 absorption (JtCO2). Addition of the diethylstilbene 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a concentration of 10(-4) M or replacement of bath chloride by gluconate reduced JtCO2 by approximately 50%, whereas replacement of luminal chloride increased JtCO2 by 40%. The carbonic anhydrase inhibitors acetazolamide and ethoxyzolamide in concentrations of 10(-4) M had little effect on JtCO2. In a concentration of 10(-3) M, acetazolamide reduced JtCO2 by only 31%. OMCTos obtained from rabbits with ammonium chloride-induced metabolic acidosis did not have increased rates of total CO2 absorption compared with the control, but treatment of animals with mineralocorticoids increased JtCO2. These results indicate that OMCTos are capable of significant bicarbonate absorption in vitro. This absorption 1) is independent of sodium transport, 2) appears to require, at least in large part, HCO3- or OH- -Cl- exchange across the basolateral cell membrane of acid-secreting cells, 3) is much more resistant to inhibition by carbonic anhydrase inhibitors than reported previously for other rabbit nephron segments, and 4) is stimulated by prior mineralocorticoid treatment of animals but not by prior metabolic acidosis in vivo.

Effect of potassium depletion and protein intake in vivo on renal tubular bicarbonate transport in vitro.

These studies evaluated the effect of decreased dietary potassium and increased dietary protein content in rabbits on systemic acid-base parameters, urine pH, and segmental renal tubular bicarbonate handling in vitro. Animals fed a potassium-deficient high-protein diet (KD) for 2 wk developed metabolic acidosis (arterial blood pH 7.31 vs. 7.43) and excreted a more acid urine than control animals fed a diet with normal amounts of potassium and protein; whereas, animals fed the same potassium deficient diet to which supplemental potassium was added (KD + K+) excreted an even more acid urine and had a blood pH of 7.36. In superficial proximal convoluted and straight tubules, there were no differences in rates of fluid and total CO2 absorption between control and KD tubules. Cortical collecting tubules obtained from KD and KD + K+ animals absorbed, and control tubules secreted total CO2 in vitro. With an ambient potassium concentration of 2.5 mM, collecting tubules obtained from the inner stripe of the outer medulla of KD animals absorbed significantly less total CO2 than control tubules. The same tendency was observed in collecting tubules from the outer stripe of the outer medulla. Plasma aldosterone levels fell with development of potassium depletion. Administration of deoxycorticosterone for 1 day to KD animals was associated with a lower urine pH and higher arterial blood pH than in untreated KD animals and with increased total CO2 absorption by both cortical collecting tubules and tubules from the inner stripe of the outer medulla.(ABSTRACT TRUNCATED AT 250 WORDS)

Femorotibial vein bypass in a child.

This report details the course and management of a 5-year-old girl with intermittent claudication, a chronic leg ulcer, and discrepancy in leg growth caused by unilateral femoropopliteal arterial occlusive lesions of obscure etiology. Angiographic evaluation, pathologic observations, and surgical management of this child are detailed. A 4-year follow-up has provided gratifying results that justify the surgical approach to this unique patient.

In vivo and in vitro studies of urinary concentrating ability in potassium-depleted rabbits.

The factors responsible for the urinary concentrating defect associated with the potassium-depleted (KD) state are uncertain. The present studies were designed to, first, determine whether a urinary concentrating defect exists in potassium-depleted rabbits and, second, to use the technique of in vitro perfusion to evaluate directly the antidiuretic hormone (ADH) responsiveness of cortical collecting tubules (CCT) in this setting. Feeding female New Zealand White rabbits a potassium-deficient diet for 2 wk caused a significant fall in plasma potassium levels in both the ad-libitum and controlled water intake groups (P less than 0.001). Muscle potassium content after 2 wk of potassium restriction fell from 45.6 +/- 0.9 to 29.0 +/- 1.2 meq/100 g fat-free dry solids (P less than 0.001). Renal papillary sodium content fell significantly from a control value of 234.6 +/- 8.0 to 182.46 +/- 10.0 meq/kg H2O after 2 wk of potassium restriction. Maximal urinary osmolality measured after 12 h of dehydration and 1.25 U pitressin IM was significantly decreased in rabbits after 2 wk of potassium restriction in both the ad-libitum and controlled water intake groups (P less than 0.001). The relationship between plasma potassium concentration and maximum urinary osmolality was significantly correlated in both the ad-libitum and controlled water intake groups, r = 0.73 and 0.68 (P less than 0.001), respectively. In addition, refeeding KD rabbits with normal chow for 1 wk resulted in normalization of both plasma potassium levels and urinary concentrating ability. CCT from control and KD rabbits were perfused in vitro at 25 degrees C. The hydraulic conductivity coefficient, Lp, was significantly reduced at all doses of ADH tested in tubules from KD rabbits when compared with control tubules. In addition, the maximal hydraulic conductivity in tubules from KD rabbits when tested with 200 microU/ml ADH at 37.5 degrees C was only 23% of control values (P less than 0.05). Furthermore, this reduced ADH responsiveness persisted when the bath potassium was elevated from 5 to 20 mM. The reflection coefficient for NaCl when compared with raffinose was 0.91 in tubules from KD animals. Thus, these data suggest that the ADH-resistant urinary concentrating defect associated with potassium depletion is due, at least in part, to a diminished responsiveness of the CCT to ADH. Therefore, further studies were designed to investigate the cellular steps involved in this abnormal response. There was no difference in the 8-para-chlorophenylthio cyclic AMP induced hydroosmotic response between CCT from KD and control rabbits. Since the cAMP-induced hydroosmotic response was similar between KD and control CCT, experiments were performed to evaluate the contribution of phosphodiesterase (PDIE) activity by using the potent PDIE inhibitor isobutylmethylxanthine (10(-4) and 10(-3)M) in the presence of ADH (200 U/ml). Although Lp was increased by PDIE inhibition in CCT from both control and KD animals, the overall hydroosmotic response in CCT from KD rabbits was still significantly reduced when compared with controls. The final experiments used forskolin to evaluate further the adenylate cyclase complex. The resulting hydroosmotic response in CCT from KD rabbits was almost identical to that obtained in controls. In conclusion, these data suggest that the decreased responsiveness of CCT from KD rabbits to ADH involves a step at or proximal to the stimulation of the catalytic subunit of adenylate cyclase, and that PDIE activity makes no contribution to this abnormal hydroosmotic response.

The effect of electronic fetal monitoring on the incidence of cesarean section.

In an attempt to elucidate the effect of electronic fetal monitoring on cesarean section rates, a review was made of the experience before and that during the 7 years after the initiation of fetal monitoring at Vanderbilt University Hospital. When a comparison was made of the cesarean section rates in the 3 years prior to fetal monitoring with those over the subsequent 2 years, no change was observed, thus indicating that, in experienced hands, a liberal use of fetal monitoring in low-risk and high-risk patients does not necessarily cause a rise in the overall incidence of cesarean sections. During this time, the neonatal mortality rate fell approximately fourfold. On the basis of this study, the belief is that a liberal attitude toward delivery by cesarean section for patients with breech presentations and other high-risk conditions contributed more to the rise in cesarean sections at Vanderbilt University Hospital than did electronic fetal monitoring. With proper education of the clinician and correct interpretation of the findings, electronic fetal monitoring need not increase cesarean section rates, but rather should allow for a more accurate description of intrapartum life.

Prostaglandin E2 vaginal suppositories in pregnancy with an anencephalic fetus.

Three patients with anencephalic fetuses were delivered vaginally following use of prostaglandin E2 (PGE2) vaginal suppositories with minimal side effects and minimal delay until delivery, even though the cervix was unfavorable. One patient had laminaria inserted 12 hours prior to a single PGE2 suppository. The fetuses were stillborn, although all had had fetal heart tones when the first suppository was inserted. The observed uterine tachysystole and hypertonus under these circumstances may be advantageous if they prevent the additional emotional stress involved with the birth of a "live" anencephalic infant.