Biomedical subjects

D S Shewach

Publications and source records attributed to D S Shewach.

At least 19 recordsLinked to original sources

PubMed1999

Cytotoxicity and accumulation of ganciclovir triphosphate in bystander cells cocultured with herpes simplex virus type 1 thymidine kinase-expressing human glioblastoma cells.

The ability of herpes simplex virus type 1 thymidine kinase (HSV-TK)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-TK-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs through the transfer of phosphorylated GCV, there is little direct proof that bystander cells can accumulate GCV nucleotides. We have studied the ability of U251 human glioblastoma cells expressing HSV-TK (U251tk cells) to induce cytotoxicity in neighboring U251 bystander cells that lack the viral kinase (U251beta gal cells) and evaluated whether this bystander cell killing is mediated by GCV nucleotides. The cytotoxicity studies demonstrated that the ratio of HSV-TK-expressing cells:bystander cells was important in determining the sensitivity of both cell types to GCV. U251tk cells cocultured with an equal number of U251beta gal cells (a 50:50 ratio) exhibited a sensitivity to GCV similar to that observed in the absence of bystander cells, with >99.8% cell kill at 1 microm GCV. However, in cultures with 10% U251tk cells and 90% bystander cells (a 10:90 ratio), 1 microM GCV decreased the survival of U251tk cells by only 54%. Strong bystander cell killing was observed at both ratios. In a 50:50 coculture of U251tk and U251beta gal cells, the survival of bystander cells was decreased by >99.5% with 3 microM GCV, whereas 30 microM GCV was required to effect a similar decrease in bystander cell survival when 90% of the culture consisted of U251beta gal cells. To determine whether this bystander cell killing may be mediated by GCV nucleotides, we developed a technique to separate the two cell populations after coculture. A U251 bystander cell line was developed from the parental cell line by transfection with the cDNA coding for green fluorescent protein (U251gfp cells), which permitted the separation of U251gfp cells from nonfluorescing U251tk cells by flow cytometry with cell sorting. With this technique, bystander cells were isolated in a viable state with >97% purity within 1 h after harvest, permitting analysis of the nucleotide pools for the presence of phosphorylated GCV. The results demonstrated that significant levels of the triphosphate of GCV (GCVTP) accumulated in bystander cells within 4 h of coculture, and this accumulation was dependent upon the percentage of HSV-TK-expressing cells as well as the concentration of GCV and the length of incubation. The proportion of GCVTP in bystander cells was consistently 50-80% of that in HSV-TK-expressing cells in the 50:50 or 10:90 cocultures, suggesting a facile transfer of phosphorylated GCV. However, the actual amount of GCVTP was as much as 8-fold lower in both the U251tk and U251beta gal cells cocultured at a ratio of 10:90 compared to those cocultured at a ratio of 50:50, which is consistent with the lesser effect on cell survival. When U251tk and U251gfp cells were cultured with 1-beta-D-arabinofuranosylthymine (araT), the 5'-triphosphate of araT accumulated in the bystander cells, demonstrating that the transfer of phosphorylated compounds between these cell types is not restricted to GCV nucleotides. However, the proportion of araT-5'-triphosphate in bystander cells compared to that in HSV-TK-expressing cells was lower than that for GCVTP, and the amount was not sufficient to decrease survival in the bystander population.

Arabinonucleotides↗

PubMed1999

Comparison of the mechanism of cytotoxicity of 2-chloro-9-(2-deoxy-2- fluoro-beta-D-arabinofuranosyl)adenine, 2-chloro-9-(2-deoxy-2-fluoro- beta-D-ribofuranosyl)adenine, and 2-chloro-9-(2-deoxy-2,2-difluoro- beta-D-ribofuranosyl)adenine in CEM cells.

In an effort to understand biochemical features that are important to the selective antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine [Cl-F( upward arrow)-dAdo], we evaluated the biochemical pharmacology of three structurally similar compounds that have quite different antitumor activities. Cl-F( upward arrow)-dAdo was 50-fold more potent as an inhibitor of CEM cell growth than were either 2-chloro-9-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)adenine [Cl-F( downward arrow)-dAdo] or 2-chloro-9-(2-deoxy-2, 2-difluoro-beta-D-ribofuranosyl)adenine [Cl-diF( upward arrow downward arrow)-dAdo]. The compounds were similar as substrates of deoxycytidine kinase. Similar amounts of their respective triphosphates accumulated in CEM cells, and the rate of disappearance of these metabolites was also similar. Cl-F( upward arrow)-dAdo was 10- to 30-fold more potent in its ability to inhibit the incorporation of cytidine into deoxycytidine nucleotides than either Cl-F( downward arrow)-dAdo or Cl-diF( upward arrow downward arrow)-dAdo, respectively, which indicated that ribonucleotide reductase was differentially inhibited by these three compounds. Thus, the differences in the cytotoxicity of these agents toward CEM cells were not related to quantitative differences in the phosphorylation of these agents to active forms but can mostly be accounted for by differences in the inhibition of ribonucleotide reductase activity. Furthermore, the inhibition of RNA and protein synthesis by Cl-F( downward arrow)-dAdo and Cl-diF( upward arrow downward arrow)-dAdo at concentrations similar to those required for the inhibition of DNA synthesis can help explain the poor antitumor selectivity of these two agents because all cells require RNA and protein synthesis.

Adenine Nucleotides↗

PubMed1998

Superior cytotoxicity with ganciclovir compared with acyclovir and 1-beta-D-arabinofuranosylthymine in herpes simplex virus-thymidine kinase-expressing cells: a novel paradigm for cell killing.

Enzyme-prodrug therapy using ganciclovir and herpes simplex virus-thymidine kinase (HSV-TK) has demonstrated excellent antitumor activity in many different types of malignant cells. Previously, we noted that ganciclovir was substantially more cytotoxic than other HSV-TK substrates. Therefore, we embarked on a study to determine the basis for the superior cytotoxicity of ganciclovir. In U251tk human glioblastoma cells that stably express HSV-TK, ganciclovir elicited a >4 log cell kill instead of the < or =1.5 log cell kill mediated by two other HSV-TK substrates, 1-beta-D-arabinofuranosylthymine (araT) and acyclovir. Study of the metabolism of these drugs demonstrated that acyclovir was poorly phosphorylated to its active triphosphate with DNA incorporation below the limit of detection, which may explain the < 1 log cell kill in these cells. Lower levels of ganciclovir triphosphate accumulated compared with araT triphosphate (araTTP) under conditions that induced < or =1 log cell kill (67 versus 1235 pmol/10(7) cells, respectively), and the half-life for the triphosphate of ganciclovir was shorter than that of araT (terminal half-lives of 15 and 41 h, respectively). Incorporation of ganciclovir monophosphate into DNA was less than that of araT monophosphate, and both analogues were retained in DNA for > or =48 h. Thus, the superior cytotoxicity of ganciclovir was not due to enhanced metabolism to active forms. Highly cytotoxic concentrations of ganciclovir produced only weak inhibition of DNA synthesis. This allowed cells to proceed through S and G2-M phases during and after drug exposure, resulting in a doubling of cell number by 48 h after drug washout. As they attempted to progress through the cell cycle a second time, ganciclovir-treated cells accumulated in early S-phase and remained there until cell death, suggesting that ganciclovir incorporation in the DNA template was important for cytotoxicity. In contrast, strong inhibition of DNA synthesis by araTTP prevented cells from traversing the cell cycle for at least 12 h after drug washout, when the active metabolite was largely degraded araT-treated cells were unable to divide for at least 72 h after drug exposure, at which point the surviving cells displayed a normal cell cycle distribution pattern. Based on the results presented here, we propose a novel paradigm in which the ability of ganciclovir to incorporate into DNA without inhibiting progression through S-phase, combined with high cytotoxicity for incorporated ganciclovir monophosphate, produces multilog cytotoxicity.

Acyclovir↗

PubMed1998

Differential ganciclovir-mediated cytotoxicity and bystander killing in human colon carcinoma cell lines expressing herpes simplex virus thymidine kinase.

The two human colon carcinoma cell lines HT-29 and SW620, which stably express herpes simplex virus thymidine kinase (HSV-TK), are sensitized to the cytotoxic effects of the antiviral drug ganciclovir (GCV). Compared with HT-29 cells, SW620 cells were more sensitive to lower GCV concentrations (<1 microM), accumulated GCV triphosphate more rapidly, and incorporated higher levels of GCV into DNA. Following a 24-hr exposure to 10 microM GCV, bystander killing was as much as sixfold greater in SW620 cells than HT-29 cells. This bystander effect was dependent on the level of HSV-TK expression, the number of cells expressing HSV-TK, and the overall confluency of the cells. However, bystander killing did not correlate with gap junctional intercellular communication as determined by microinjection of Lucifer Yellow fluorescent dye. SW620 cells were coupled to <3% adjacent cells (compared with >50% for HT-29 cells), but were still able to transfer phosphorylated GCV to bystander cells as soon as 4 hr after drug was added. These results emphasize the importance of cell-specific metabolism in HSV-TK/GCV-mediated cytotoxicity and may suggest a novel mechanism for bystander killing.

Adenoviridae↗

PubMed1998

Metabolism in human cells of the D and L enantiomers of the carbocyclic analog of 2'-deoxyguanosine: substrate activity with deoxycytidine kinase, mitochondrial deoxyguanosine kinase, and 5'-nucleotidase.

The carbocyclic analog of 2'-deoxyguanosine (CdG) has broad-spectrum antiviral activity. Because of recent observations with other nucleoside analogs that biological activity may be associated the L enantiomer rather than, as expected, with the D enantiomer, we have studied the metabolism of both enantiomers of CdG to identify the enzymes responsible for the phosphorylation of CdG in noninfected and virally infected human and duck cells. We have examined the enantiomers as substrates for each of the cellular enzymes known to catalyze phosphorylation of deoxyguanosine. Both enantiomers of CdG were substrates for deoxycytidine kinase (EC 2.7.1.74) from MOLT-4 cells, 5'-nucleotidase (EC 3.1.3.5) from HEp-2 cells, and mitochondrial deoxyguanosine kinase (EC 2.7.1.113) from human platelets and CEM cells. For both deoxycytidine kinase and mitochondrial deoxyguanosine kinase, the L enantiomer was the better substrate. Even though the D enantiomer was the preferred substrate with 5'-nucleotidase, the rate of phosphorylation of the L enantiomer was substantial. The phosphorylation of D-CdG in MRC-5 cells was greatly stimulated by infection with human cytomegalovirus. The fact that the phosphorylation of D-CdG was stimulated by mycophenolic acid and was not affected by deoxycytidine suggested that 5'-nucleotidase was the enzyme primarily responsible for its metabolism in virally infected cells. D-CdG was extensively phosphorylated in duck hepatocytes, and its phosphorylation was not affected by infection with duck hepatitis B virus. These results are of importance in understanding the mode of action of D-CdG and related analogs and in the design of new biologically active analogs.

5'-Nucleotidase↗

PubMed1997

Improved method to prepare RNA-free DNA from mammalian cells.

To isolate DNA for nucleoside analog incorporation studies, many investigators use RNase A to remove RNA from total cellular nucleic acid. We observed persistence of ribonucleotides from RNA in nucleic acid samples treated with RNase A alone. Although incubation of [5-3H]uridine-labeled nucleic acid with 50 microg/ml RNase A decreased tritium by 97%, HPLC analysis of the resulting DNA preparation digested to nucleosides revealed high levels of ribonucleosides. Increasing RNase A 10-fold (500 microg/ml) effected only a 1.7-fold reduction in ribonucleosides. Overall, the level of ribonucleosides was one-fourth that of the deoxynucleosides, primarily due to the high levels of guanosine. It was hypothesized that the ribonucleosides originated from guanosine-rich tracts of RNA since RNase A cuts preferentially 3' to pyrimidine monophosphates and to some extent after AMP. The addition of 0.05 microg/ml RNase T1, which preferentially cleaves RNA 3' to GMP, decreased total ribonucleosides by nearly 20-fold. In conclusion, we have developed a rapid method which removes greater then 99% of cellular RNA from nucleic acid extracts and a reversed-phase HPLC procedure that detects RNA contamination more sensitively than [5-3H]uridine labeling. These methods are useful for the determination of analog incorporation into DNA, especially for agents which incorporate into both DNA and RNA.

Animals↗

PubMed1997

Kinetic analysis of human deoxycytidine kinase with the true phosphate donor uridine triphosphate.

Deoxycytidine kinase is the rate-limiting process in the activation for several clinically important antitumor agents. Previous studies have focused on deoxycytidine (dCyd) and adenosine triphosphate (ATP) as substrates for this enzyme. In view of recent data indicating that uridine triphosphate (UTP) is the physiologic phosphate donor for this enzyme, a study of the kinetic properties of dCyd kinase with dCyd and UTP was undertaken. The results presented here demonstrate that UTP and ATP produce kinetically distinguishable differences in nucleoside phosphorylation by dCyd kinase. At high dCyd concentrations, dCyd kinase exhibited substrate activation with ATP. In contrast, in the presence of UTP, substrate inhibition was observed at concentrations of dCyd greater than 3 microM. Inhibition by dCyd was noncompetitive with respect to UTP and could not be reversed by a 200-fold increase in UTP concentration, indicating that the inhibition was not due to dCyd binding at the nucleotide binding site. The kinetic mechanism for dCyd kinase was determined with dCyd and UTP as substrates. UTP was the preferred phosphate donor with a true Km value of 1 microM compared to 54 microM with ATP, resulting in a 50-fold greater substrate efficiency for UTP. Although the double-reciprocal plots with UTP produced parallel lines, initial velocity plots with other phosphate donors and product inhibition studies indicated that dCyd kinase formed a ternary complex with its substrates. The parallel lines with UTP were apparently due to a low dissociation constant for UTP, which was calculated as more than 13-fold lower than its Km value. Analysis of product inhibition studies indicated that dCyd kinase followed an ordered A-B random P-Q reaction sequence, with UTP as the first substrate to bind. In contrast, previous results demonstrated a random bi-bi sequence for dCyd kinase in the presence of ATP. The combined results indicate that the enzyme can follow a random bi-bi reaction sequence, but with UTP as the phosphate donor, the addition of nucleotide prior to dCyd is strongly preferred. The noncompetitive substrate inhibition, which was independent of UTP concentration, indicates that high concentrations of dCyd promote addition of the nucleoside prior to UTP, resulting in a lower velocity.

Adenosine Triphosphate↗

PubMed1997

Gemcitabine-mediated radiosensitization.

Gemcitabine is a potent radiosensitizer of human tumor cells. This review summarizes our preclinical and early clinical studies designed to elucidate the mechanism of action of gemcitabine and phase I trials conducted to determine the optimal dose and schedule. Gemcitabine was found to radiosensitize a wide variety of human tumor cells in culture, particularly cells derived from cancers of the pancreas, breast, and head and neck. Radiosensitization occurs under conditions in which cells demonstrate concurrent redistribution into S phase and deoxyadenosine triphosphate pool depletion. These conditions can be produced by either a long (24-hour) exposure to a low concentration of gemcitabine (10 nmol/L) or by a brief (2-hour) treatment with higher but clinically relevant concentrations (100 nmol/L to 3 micromol/L). Under the latter conditions, sensitization can be detected 4 hours after treatment and last for up to 2 days. These preclinical data were useful in the design of a gemcitabine dose escalation trial in combination with standard radiation for patients with unresectable head and neck cancer. Although this trial is not yet complete, the starting dose of gemcitabine, which is far below the maximum tolerated dose for the drug when used alone, significantly potentiates the toxicity of radiation treatment. We conclude that gemcitabine is a promising radiation sensitizer that needs to be developed cautiously if excessive normal tissue toxicity is to be avoided.

Carcinoma, Squamous Cell↗

PubMed1997

Delayed radiosensitization of human colon carcinoma cells after a brief exposure to 2',2'-difluoro-2'-deoxycytidine (Gemcitabine).

We have shown that 2',2'-difluoro-2'-deoxycytidine (dFdCyd; Gemcitabine), a deoxycytidine analogue, is a potent radiation sensitizer when cells are exposed to it continuously for >16 h in low concentrations (in the range of 10 nM). However, the most common method of clinical administration is by short-term infusion (30-90 min). Therefore, we wished to determine under what conditions dFdCyd could produce radiosensitization after a relatively brief exposure to drug. We hypothesized that the long half-life of the phosphorylated metabolites of dFdCyd would produce long-lasting dNTP pool perturbation, particularly dATP pools, leading to radiosensitization hours or even days after the drug was removed from the medium. We tested this hypothesis by exposing HT29 human colon cancer cells for 2 h to clinically relevant concentrations of dFdCyd, removing the drug from the medium, and assessing radiation sensitivity up to 72 h later. We found that 100 nM dFdCyd, which was noncytotoxic, radiosensitized HT29 cells up to 48 h after drug removal. During this period, there was an increase in the S phase population, whereas by 72 h after drug removal, the cell cycle distribution resembled that seen under control conditions. dATP pools remained depleted throughout the 72-h period after drug treatment. This study supports the hypothesis that radiosensitization occurs in cells that are replicating DNA in the presence of perturbed dNTP pools. Furthermore, they may be useful in the design of rational clinical trials using dFdCyd as a radiation sensitizer.

Antimetabolites, Antineoplastic↗

PubMed1996

Radiosensitizing nucleosides.

Chemotherapeutic drugs that perturb nucleotide metabolism have the potential to produce substantial sensitization of tumor cells to radiation treatment. The process is called radiosensitization, and the agents that induce it are called radiosensitizers. The clinical effectiveness of fluoropyrimidines as radiosensitizers has been proven in multiple randomized trials. Thymidine analogues and hydroxyurea also appear to produce clinically relevant increases in radiation sensitivity. Recent laboratory investigations have identified difluorodeoxycytidine (gemcitabine) and fludarabine as promising agents to use in combination with radiation. Until recently, little was known about how the biochemical changes caused by these drugs produced radiosensitization. However, advances in related fields, such as cell cycle checkpoint control, have permitted the development of a hypothesis that may explain the relative tumor selectivity of fluoropyrimidine-mediated radiosensitization. In addition, recent findings suggest that the rational manipulation of drug administration schedules and the use of combinations of radiosensitizers have the potential to improve the efficacy of the currently used agents and to establish the benefit of new ones.

Animals↗

PubMed1996

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression in the central nervous system of HPRT-deficient mice following adenoviral-mediated gene transfer.

In this study we show that recombinant adenovirus can augment hypoxanthine-guanine phosphoribosyltransferase (HPRT) levels in the central nervous system (CNS) of HPRT-deficient mice. Recombinant adenovirus containing the cDNA for rat HPRT (rHPRT) expressed from the Rous sarcoma virus LTR (RSV LTR) was constructed (AdRSVrHPRT). AdRSVrHPRT was injected into the right caudate nucleus of 7-week-old HPRT-deficient mice. Brains were analyzed for gene transfer, transgene expression and function by DNA PCR, in situ RNA hybridization, and enzyme bioactivity. The results show that rHPRT cDNA delivered by an adenoviral vector can augment HPRT levels in brain tissue and documents the utility of gene transfer to restore HPRT activity in an HPRT-deficient CNS.

Adenoviridae↗

PubMed1996

Preclinical studies of chemotherapy and radiation therapy for pancreatic carcinoma.

The use of radiation therapy combined with 5-fluorouracil (5-FU) in the treatment of pancreatic cancer has been well established. It has been hypothesized that any benefit from combined 5-FU and radiation has been due to radiosensitization. Improved therapy could result from a better understanding of the mechanism of radiosensitization and the development of compounds capable of providing better radiosensitization. This article reviews preclinical findings on the mechanism of cytotoxicity and radiosensitization for 5-FU, fluorodeoxyuridine, thymidine analogs, and gemcitabine (2',2'-difluorodeoxycytidine) and discusses the clinical implications of these findings.

Antimetabolites, Antineoplastic↗

PubMed1996

Radiosensitization of pancreatic cancer cells by 2',2'-difluoro-2'-deoxycytidine.

PURPOSE: We have reported that the deoxycytidine analog 2',2'difluoro-2'-deoxycytidine (dFdCyd) is a potent radiosensitizer of HT29 human colon cancer cells probably through its effects on intracellular deoxyribonucleotide (dNTP) pools. Because dFdCyd has activity against pancreatic cancer in clinical trials, we wished to determine if dFdCyd would radiosensitize human pancreatic cancer cells. METHODS AND MATERIALS: We assessed the effect of dFdCyd on radiation sensitivity of two human pancreatic cancer cell lines, Panc-1 and BxPC-3. To begin to investigate the mechanism of sensitization, we determined the effect of dFdCyd on dNTP pools and cell cycle distribution. RESULTS: We found that dFdCyd produced radiation enhancement ratios of 1.7-1.8 under noncytotoxic conditions in both cell lines. Sensitization was not associated with intracellular levels of 2',2'-difluoro-2'-deoxycytidine triphosphate, the cytotoxic metabolite of dFdCyd, but occurred when dATP pools were depleted below the level of approximately 1 micromolar. Although both cell lines showed substantial cell cycle redistribution after drug treatment, the flow cytogram of the BxPC-3 cells would not, by itself, be anticipated to result in increased radiation sensitivity. CONCLUSIONS: These findings demonstrate that dFdCyd is a potent radiation sensitizer of human pancreatic cancer cells and support the development of a clinical protocol using combined dFdCyd and radiation therapy in the treatment of pancreatic cancer.

Adenosine Triphosphate↗

PubMed1996

Gemcitabine and radiosensitization in human tumor cells.

Gemcitabine is a nucleoside analogue with excellent clinical activity against solid tumors. Within the cell, gemcitabine is rapidly phosphorylated to its active di- and triphosphate metabolites. Cytotoxicity with gemcitabine appears to be related to multiple effects on DNA replication, where gemcitabine triphosphate can serve as both an inhibitor and substrate for DNA synthesis. Gemcitabine diphosphate inhibits ribonucleotide reductase, producing decreases in cellular dNTP pool levels in a cell-specific manner. These two major characteristics of gemcitabine, reduction in cellular dNTP pools and incorporation into DNA, are features of other antimetabolites antitumor agents which also exhibit radiosensitizing properties. Based on these favorable metabolic characteristics and the clinical activity of gemcitabine in tumor types which are commonly treated with radiation, the ability of gemcitabine to enhance X-radiation induced cytotoxicity was evaluated. Gemcitabine has been shown to be a potent radiosensitizer in a variety of tumor cell lines, including HT-29 colorectal carcinoma, pancreatic cancer, breast, non-small cell lung and head and neck cancer cell lines. Gemcitabine was most effective as a radiosensitizer when administered at least 2 hours prior to irradiation. For most cell lines, radiosensitization was evident at non-cytotoxic concentrations. The extent of radiosensitization increased with both increasing gemcitabine concentration and duration of exposure. Radiosensitization did not require redistribution of cells into a more radiosensitive phase of the cell cycle. The major metabolic effects observed under radiosensitizing conditions were the accumulation of high levels of gemcitabine triphosphate, and a selective decrease in the cellular dATP pool. The pattern of dATP decrease paralleled the increase in radiosensitization, whereas the level of gemcitabine triphosphate was not associated with the enhanced sensitivity to radiation. Compared to other radiosensitizers, the advantage of gemcitabine is that is can induce radiosensitization at concentrations that are 1000 times lower than typical plasma levels obtained with this drug. These studies will be used as guidelines for developing clinical trials of gemcitabine with radiation.

Antimetabolites, Antineoplastic↗

PubMed1996

Paracrine mediation of calcium signaling in human SK-N-MCIXC neuroepithelioma cells.

Paracrine-mediated Ca2+ signaling in SK-N-MCIXC neuroepithelioma cells was evaluated by means of two experimental paradigms. In the first, single SK-N-MCIXC cells were microinjected with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and cytoplasmic Ca2+ was monitored by fura 2 digital-imaging microfluorometry. In response to Ins(1,4,5)P3 or CaCl2, but not inositol 1,3,4-trisphosphate, an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) was observed in injected cells and also in neighboring cells. The direction of intercellular propagation of Ca2+ signals was influenced by the presence of a flow in the extracellular medium and occurred in the absence of any detectable gap-junctional communication. The P2 purinoceptor antagonist suramin, but not antagonists of other phosphoinositide-linked receptors, blocked cell-to-cell Ca2+ signaling initiated by microinjections of Ins(1,4,5)P3. In the second paradigm, conditioned medium (CM) obtained from monolayers of SK-N-MCIXC cells elicited increases in [Ca2+]i when reapplied to cells on coverslips. The Ca(2+)-mobilizing activity of CM was reversibly antagonized by suramin and abolished by pretreatment with apyrase. The presence of nucleotide di- and triphosphates in CM was confirmed by high-performance liquid chromatography. We conclude that SK-N-MCIXC cells release nucleotides that then activate specific receptors on neighboring cells. A rise in [Ca2+]i in these cells, and subsequent additional release of nucleotides, serves to further the propagation of intercellular Ca2+ signals.

Biological Transport↗

PubMed1996

Radiosensitization of human solid tumor cell lines with gemcitabine.

Gemcitabine has high clinical activity in several solid tumors that are treated with radiotherapy and/or chemotherapy. The mode of action of gemcitabine involves a number of intracellular changes that are shared by other radiation sensitizing anticancer drugs. There is therefore a clear rationale for investigating the interaction between gemcitabine and radiation in human cancer cell lines in vitro. Gemcitabine has been shown to be a potent radiosensitizer in human colorectal, pancreatic, and other solid tumor cell lines. Gemcitabine produces radiation enhancement ratios that are higher than those of other established radio-sensitizing agents, and this radiation enhancement occurs at low noncytotoxic concentrations. Radiosensitization increases with dose and with duration of exposure to gemcitabine, and is greatest when exposure to gemcitabine precedes radiation. The primary radiosensitizing effect of gemcitabine seems to be associated with depletion of endogenous nucleotide pools; deoxyadenosine triphosphate reduction is particularly striking, even at the lowest concentrations of gemcitabine. These studies suggest a number of approaches that are relevant to the clinical use of gemcitabine in patients with solid tumors.

Antimetabolites, Antineoplastic↗

PubMed1995

Radiosensitization of human tumor cells by gemcitabine in vitro.

Gemcitabine is a novel nucleoside analog that has shown clinical activity in a variety of solid tumors. The use of gemcitabine in combination with radiation was investigated in vitro using HT-29 colon carcinoma cells. Gemcitabine was demonstrated to be a potent radiosensitizer even at noncytotoxic concentrations. Increasing the concentration of gemcitabine and/or the duration of exposure increased the radiosensitization of cells. Biochemical studies indicated that a reduction in the dATP pool was the most likely mechanism of radiosensitization by gemcitabine. This effect is of great clinical importance and should be investigated in vivo.

Cell Cycle↗

PubMed1994

Metabolism of 2',2'-difluoro-2'-deoxycytidine and radiation sensitization of human colon carcinoma cells.

Difluorodeoxycytidine (dFdCyd) is a new antimetabolite with clinical activity in patients with solid tumors but not leukemias. We have studied the metabolism, cytotoxicity, and radiosensitizing properties of dFdCyd in HT-29 human colon carcinoma cells. The results demonstrated that dFdCyd rapidly accumulated as the 5'-triphosphate dFdCTP in HT-29 cells, which was eliminated slowly in the absence of dFdCyd with a half-life of > 12 h. Accumulation of dFdCTP was associated with rapid depletion of cellular dATP pools. Exposure to the concentration that reduces cell survival by 50% of 30 nM dFdCyd decreased dATP levels by > 80% within 4 h. dGTP pools were depleted at higher concentrations of dFdCyd, whereas smaller decreases were effected in dTTP and dCTP pools. These results contrast with previous reports in leukemic cells which demonstrated that dFdCyd exposure depleted the endogenous dCTP pool to a greater extent than the dTTP, dATP, or dGTP pools. Based on these data, we suggest that the profound depletion by dFdCyd of dATP and dGTP pools in HT-29 compared to leukemic cells accounts for the superiority of this agent in solid tumors versus leukemias. Additional studies demonstrated that dFdCyd was a potent radiosensitizer in HT-29 cells. Maximal radiosensitization was observed when cells were irradiated immediately following dFdCyd exposure instead of prior to or in the middle of drug treatment. Radiation sensitization was dose and time dependent, with a noncytotoxic exposure to 10 nM dFdCyd for 24 h or 30 nM dFdCyd for 16 h producing a radiation enhancement ratio of approximately 2. Under these conditions, only the cellular dATP pool was depleted. When cells were exposed to higher concentrations of dFdCyd for 4 h, equivalent radiosensitization with a radiation enhancement ratio of 1.4 was obtained using 0.1, 1.0, or 10 microM dFdCyd. Despite a 15-fold increase in dFdCTP and depletion of dGTP and dCTP pools to < 25% of the control value with 10 microM compared to 0.1 microM dFdCyd, no increase in radiosensitization was observed. These results suggest that dATP depletion is an important factor in the radiosensitizing property of this promising new antitumor compound.

Antimetabolites, Antineoplastic↗