Biomedical subjects

L Li

Publications and source records attributed to L Li.

At least 649 records · Page 36Linked to original sources

PubMed2000

Expression of the TGF-beta receptor gene and sensitivity to growth inhibition following polyamine depletion.

Our previous studies have shown that inhibition of polyamine biosynthesis increases the sensitivity of intestinal epithelial cells to growth inhibition induced by exogenous transforming growth factor-beta (TGF-beta). This study went further to determine whether expression of the TGF-beta receptor genes is involved in this process. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Administration of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (the rate-limiting enzyme for polyamine synthesis), for 4 and 6 days depleted cellular polyamines putrescine, spermidine, and spermine in IEC-6 cells. Polyamine depletion by DFMO increased levels of the TGF-beta type I receptor (TGF-betaRI) mRNA and protein but had no effect on the TGF-beta type II receptor expression. The induced TGF-betaRI expression after polyamine depletion was associated with an increased sensitivity to growth inhibition induced by exogenous TGF-beta but not by somatostatin. Extracellular matrix laminin inhibited IEC-6 cell growth without affecting the TGF-beta receptor expression. Laminin consistently failed to induce the sensitivity of TGF-beta-mediated growth inhibition. In addition, decreasing TGF-betaRI expression by treatment with retinoic acid not only decreased TGF-beta-mediated growth inhibition in normal cells but also prevented the increased sensitivity to exogenous TGF-beta in polyamine-deficient cells. These results indicate that 1) depletion of cellular polyamines by DFMO increases expression of the TGF-betaRI gene and 2) increased TGF-betaRI expression plays an important role in the process through which polyamine depletion sensitizes intestinal epithelial cells to growth inhibition induced by TGF-beta.

Activin Receptors, Type I↗

PubMed2000

Cell-specific localization of insulin-like growth factor binding protein mRNAs in rat liver.

The liver is a major source of circulating insulin-like growth factor I (IGF-I), and it also synthesizes several classes of IGF binding proteins (IGFBPs). Synthesis of IGF-I and IGFBPs is regulated by hormones, growth factors, and cytokines. They are nutritionally regulated and expressed in developmentally specific patterns. To gain insight into cellular regulatory mechanisms that determine hepatic synthesis of IGF-I and IGFBPs and to identify potential target cells for IGF-I within the liver, we studied the cellular sites of synthesis of IGF-I, IGF receptor, growth hormone (GH) receptor, and IGFBPs in freshly isolated rat hepatocytes, endothelial cells, and Kupffer cells. We also localized cellular sites of IGFBP synthesis by in situ hybridization histochemistry. Western ligand and immunoblot analyses were used to determine IGFBP secretion by isolated cells. Two IGF-I mRNA subtypes with different 5' ends (class 1 and class 2) were detected in all isolated liver cell preparations. Type 1 IGF receptor mRNA was detected in endothelial cells, indicating that these cells are a local target for IGF actions in liver. GH receptor was expressed in all cell preparations, consistent with GH regulation of IGF-I and IGFBP synthesis in multiple liver cell types. The IGFBPs expressed striking cell-specific expression. IGFBP-1 was synthesized only in hepatocytes, and IGFBP-3 was expressed in Kupffer and endothelial cells. IGFBP-4 was expressed at high levels in hepatocytes and at low levels in Kupffer and endothelial cells. Cell-specific expression of distinct IGFBPs in the liver provides the potential for cell-specific regulation of hepatic and endocrine actions of IGF-I.

Animals↗

PubMed2000

Glutathione oxidation and PTPase inhibition by hydrogen peroxide in Caco-2 cell monolayer.

The role of H(2)O(2) and protein thiol oxidation in oxidative stress-induced epithelial paracellular permeability was investigated in Caco-2 cell monolayers. Treatment with a H(2)O(2) generating system (xanthine oxidase + xanthine) or H(2)O(2) (20 microM) increased the paracellular permeability. Xanthine oxidase-induced permeability was potentiated by superoxide dismutase and prevented by catalase. H(2)O(2)-induced permeability was prevented by ferrous sulfate and potentiated by deferoxamine and 1,10-phenanthroline. GSH, N-acetyl-L-cysteine, dithiothreitol, mercaptosuccinate, and diethylmaleate inhibited H(2)O(2)-induced permeability, but it was potentiated by 1,3-bis(2-chloroethyl)-1-nitrosourea. H(2)O(2) reduced cellular GSH and protein thiols and increased GSSG. H(2)O(2)-mediated reduction of GSH-to-GSSG ratio was prevented by ferrous sulfate, GSH, N-acetyl-L-cysteine, diethylmaleate, and mercaptosuccinate and potentiated by 1,10-phenanthroline and 1, 3-bis(2-chloroethyl)-1-nitrosourea. Incubation of soluble fraction of cells with GSSG reduced protein tyrosine phosphatase (PTPase) activity, which was prevented by coincubation with GSH. PTPase activity was also lower in H(2)O(2)-treated cells. This study indicates that H(2)O(2), but not O(2)(-). or.OH, increases paracellular permeability of Caco-2 cell monolayer by a mechanism that involves oxidation of GSH and inhibition of PTPases.

Acetylcysteine↗

PubMed2000

Phosphorylation of phospholamban and troponin I in beta-adrenergic-induced acceleration of cardiac relaxation.

Activation of cAMP-dependent protein kinase A (PKA) in ventricular myocytes by isoproterenol (Iso) causes phosphorylation of both phospholamban (PLB) and troponin I (TnI) and accelerates relaxation by up to twofold. Because PLB phosphorylation increases sarcoplasmic reticulum (SR) Ca pumping and TnI phosphorylation increases the rate of Ca dissociation from the myofilaments, both factors could contribute to the acceleration of relaxation seen with PKA activation. To compare quantitatively the role of TnI versus PLB phosphorylation, we measured relaxation rates before and after maximal Iso treatment for twitches of matched amplitudes in ventricular myocytes and muscle from wild-type (WT) mice and from mice in which the PLB gene was knocked out (PLB-KO). Because Iso increases contractions, even in the PLB-KO mouse, extracellular [Ca] or sarcomere length was adjusted to obtain matching twitch amplitudes (in the presence and absence of Iso). In PLB-KO myocytes and muscles (which were allowed to shorten), Iso did not alter the time constant (tau) of relaxation ( approximately 29 ms). However, with increasing isometric force development in the PLB-KO muscles, Iso progressively but modestly accelerated relaxation (by 17%). These results contrast with WT myocytes and muscles where Iso greatly reduced tau of cell relaxation and intracellular Ca concentration decline (by 30-50%), independent of mechanical load. The Iso treatment used produced comparable increases in phosphorylation of TnI and PLB in WT. We conclude that the effect of beta-adrenergic activation on relaxation is mediated entirely by PLB phosphorylation in the absence of external load. However, TnI phosphorylation could contribute up to 14-18% of this lusitropic effect in the WT mouse during maximal isometric contractions.

Adrenergic beta-Agonists↗

PubMed2000

Conditioned immunosuppressive effect of cyclophosphamide on delayed-type hypersensitivity response and a preliminary analysis of its mechanism.

In the present study, camphor odor and intraperitoneal (i.p.) injection of cyclophosphamide (CY) were used as conditioned stimulus (CS) and unconditioned stimulus (US), respectively. In the unconditioned group, mice were exposed to camphor odor for 1 h followed by an i.p. injection of CY (75 mg/kg). On the next day, the above CS/US association trial session was repeated followed by smearing dinitrochlorobenzene (DNCB) on mouse abdominal skin for sensitizing the animal for delayed-type hypersensitivity (DTH) response. Five days after DNCB sensitization, mice were exposed to camphor odor (1 h), followed by an i.p. injection of CY, and then DNCB was smeared on the left ear of mice for the challenge of DTH response. Both the left/right ear weight ratio and the activity of leukocyte migration inhibitory factor (LMIF) were used as the index of DTH response, which was done 24 h after DNCB challenge. In the conditioned group, the treatment was the same as that in the unconditioned group, except that normal saline was injected on day 5 instead of CY. Furthermore, in order to analyze the mechanism of the conditioned response (CR), the mouse serum from the conditioned group (CR serum) was injected into normal mice 6 h prior to DNCB challenge. Results showed that in the conditioned group, left/right ear weight ratio and LMIF activity were statistically lower than that in the DTH group, and there was no difference between conditioned and unconditioned groups. Thus, an animal model of conditioned immunosuppressive response had been established. The results also showed that after CR serum was injected into normal mice, DTH response was also significantly suppressed. However, if CR serum was treated with dialysis (10,000 molecular weight cut-off), the suppressive effect of CR serum on DTH response disappeared. Taken together, the data suggested that a chemical compound(s) in serum, with a molecular weight less than 10,000, was important in mediating the conditioned immunosuppressive response. This may be a very important molecule(s) that could be very critical to our understanding of the interaction between the central nervous system and immune function.

Animals↗

PubMed2000

Regulation of acyl-coenzyme A:cholesterol acyltransferase (ACAT) synthesis, degradation, and translocation by high-density lipoprotein(2) at a low concentration.

(,Although plasma HDL(2) cholesterol concentration stands in inverse relation to risk for atherosclerotic disease, little is known about the mechanism of the apparent cardioprotection. In mouse P388D1 macrophages, HDL(2) at a low concentration (< or = 40 microg/mL) inhibits macrophage acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyzes esterification of intracellular cholesterol. The effects of HDL(2) on ACAT synthesis, degradation, and intracellular translocation were investigated in mouse P388D1 macrophages. HDL(2) at a low concentration enhanced ACAT synthesis but not total ACAT mass. Immunocytochemical studies showed that in the absence of lipoproteins, ACAT associated primarily with the perinuclear region of the cell. The addition of HDL(2), however, induced the transfer of ACAT to vesicular structures and the cell periphery adjacent to the plasma membrane. Subfractionation combined with immunoprecipitation complemented these observations and showed that HDL(2) promoted the transfer of ACAT to the plasma membrane fraction. Brefeldin A, which inhibits vesicular protein transport from the endoplasmic reticulum to the Golgi compartment in mammalian cells, blocked ACAT translocation and partially restored ACAT activity. These results suggest that HDL(2) is an initiating factor in a signal transduction pathway that leads to intracellular ACAT translocation and inactivation.

Animals↗

PubMed2000

Can a brief clinical practicum influence physicians' communications with patients about alcohol and drug problems? Results of a long-term follow-up.

BACKGROUND: A survey was conducted in 1996 to assess the perceptions of medical school graduates concerning alcohol and drug problems among their patients, and their attitudes and comfort toward addressing these issues. Survey questionnaires were sent to all individuals who graduated from Wright State University School of Medicine, Dayton, Ohio, between 1982 and 1990. PURPOSE: The study aimed to assess the long-term impact that participation in a brief clinical experience in tandem with a standard didactic substance abuse curriculum material had on former medical students now in practice. The study examined how these physicians perceived their skills in communicating with their patients about alcohol and drug use, and attendant problems. METHODS: An Alcohol and Drug Use Communication (ADUC) scale was developed by combining several survey items. Data analyses included correlation assessments of the ADUC scale and other variables, and multiple-regression analyses for identifying factors independently associated with the ADUC scale although other factors were controlled. RESULTS: Former students who participated in the brief clinical program, known as the Weekend Intervention Program, as part of their medical school curriculum were more likely to report having better communication concerning alcohol and drug use with their patients than students who only had didactic education. Also, additional training in addictions after graduation was significantly associated with better alcohol and drug use communication between physician and patient. Former students who participated in the Weekend Intervention Program were more than twice as likely as those without the experience to report confronting at least 10% of their patients about their concerns about the patients' alcohol or drug use. CONCLUSIONS: The results of this study suggest that undergraduate medical students' participation in a modest clinical program can enhance substance abuse education. Through increased training in substance abuse, physicians reported greater confidence in their ability to relate to patients with substance abuse problems.

Alcoholism↗

PubMed2000

Hrs interacts with SNAP-25 and regulates Ca(2+)-dependent exocytosis.

Synaptosome-associated protein of 25 kDa (SNAP-25) is a neuronal membrane protein essential for synaptic vesicle exocytosis. To investigate the mechanisms by which SNAP-25 mediates neurosecretion, we performed a search for proteins that interact with SNAP-25 using a yeast two-hybrid screen. Here, we report the isolation and characterization of a SNAP-25-interacting protein that is the rat homologue of mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Hrs specifically interacts with SNAP-25, but not SNAP-23/syndet. The association of Hrs and SNAP-25 is mediated via coiled-coil interactions. Using an Hrs-specific antibody, we have shown that Hrs is highly enriched in brain, where it codistributes with SNAP-25 in most brain regions. Subcellular fractionation studies demonstrate that in brain, Hrs exists in both cytosolic and membrane-associated pools. Studies using indirect immunofluorescence and confocal microscopy reveal that, in addition to early endosomes, Hrs is also localized to large dense-core secretory granules and synaptic-like microvesicles in nerve growth factor-differentiated PC12 cells. Moreover, overexpression of Hrs in PC12 cells inhibits Ca(2+)-dependent exocytosis. These results suggest that Hrs is involved in regulation of neurosecretion through interaction with SNAP-25.

Amino Acid Sequence↗

PubMed2000

Mass spectrometric survey of peptides in cephalopods with an emphasis on the FMRFamide-related peptides.

A matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) survey of the major peptides in the stellar, fin and pallial nerves and the posterior chromatophore lobe of the cephalopods Sepia officinalis, Loligo opalescens and Dosidicus gigas has been performed. Although a large number of putative peptides are distinct among the three species, several molecular masses are conserved. In addition to peptides, characterization of the lipid content of the nerves is reported, and these lipid peaks account for many of the lower molecular masses observed. One conserved set of peaks corresponds to the FMRFamide-related peptides (FRPs). The Loligo opalescens FMRFa gene has been sequenced. It encodes a 331 amino acid residue prohormone that is processed into 14 FRPs, which are both predicted by the nucleotide sequence and confirmed by MALDI MS. The FRPs predicted by this gene (FMRFa, FLRFa/FIRFa and ALSGDAFLRFa) are observed in all three species, indicating that members of this peptide family are highly conserved across cephalopods.

Amino Acid Sequence↗

PubMed2000

Symmetries of cross-polarization diffraction coefficients of gratings

In a recent paper [J. Opt. Soc. Am. A 16, 1108 (1999)] Logofatu et al. demonstrated by experimental and numerical evidence that the 0th-order cross-polarization (s to p and p to s) reflection coefficients of isotropic, symmetrical, surface-relief gratings in conical mount are identical. Here an analytical proof is given to show that the observed identity is merely a manifestation of the electromagnetic reciprocity theorem for the 0th-order diffraction of symmetrical gratings. The above result is further generalized to bianisotropic gratings, to the 0th-order cross-polarization transmission coefficients, and to the mth-order reflection and transmission coefficients when the wave vector of the incident plane wave and the negative of the wave vector of the mth reflected order are symmetrical with respect to the plane perpendicular to the grating grooves.

Journal Article↗

PubMed2000

The difference in the carboxy-terminal sequence is responsible for the difference in the activity of chicken and rat liver fructose-2,6-bisphosphatase.

The fructose-2,6-bisphosphatase domain of the bifunctional chicken liver enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase shares approximately 95% amino acid sequence homology with that of the rat enzyme. However, these two enzymes are significantly different in their phosphatase activities. In this report, we show that the COOH-terminal 25 amino acids of the two enzymes are responsible for the different enzymatic activities. Although these 25 amino acids are not required for the phosphatase activity, their removal diminishes the differences in the activities between the two enzymes. In addition, two chimeric molecules (one consisting of the catalytic core of the chicken bisphosphatase domain and the rat COOH-terminal 25 amino acids, and the other consisting of most of the intact chicken enzyme and the rat COOH-terminal 25 amino acids) showed the same kinetic properties as the rat enzyme. Furthermore, substitution of the residues Pro456Pro457Ala458 of the chicken enzyme with GluAlaGlu, the corresponding sequence in the rat liver enzyme, yields a chicken enzyme that behaves like the rat enzyme. These results demonstrate that the different bisphosphatase activities of the chicken and rat liver bifunctional enzymes can be attributed to the differences in their COOH-terminal amino acid sequences, particularly the three residues.

Amino Acid Sequence↗

PubMed2000

Neonatal tolerant immunity for vaccination against autoimmunity.

Autoimmunity arises when the immune system no longer tolerates self and precipitates lymphocyte reactivity against our own antigens. Although the developing T cell repertoire is constantly purging, self-recognition events do exist when such tight control is evaded and autoreactive lymphocytes escape the thymus (the sites of T cell development) and migrate to the periphery. Upon activation these autoreactive cells may exert aggressive behavior toward one's own tissues and organs leading to autoimmune disease. Multiple sclerosis, Rheumatoid arthritis, and type I diabetes are autoimmune diseases mediated by autoreactive T cells. A logical approach to prevent such autoimmunity would be to reprogram those lymphocytes to tolerate the self antigen. Injection of antigen at the neonatal stage promotes a state of tolerance such that successive encounter with antigen does not precipitate aggressive reactions. The mechanism underlying neonatal tolerance involves priming of T cells whose effector functions do not cause inflammatory reactions upon recognition of antigen but rather induce protective immunity. This form of tolerant immunity provides an attractive strategy for vaccination against autoimmunity. Herein, it is shown that neonatal exposure to a self-peptide-immunoglobulin chimera drives a tolerant immunity toward the self-peptide and protects against the autoimmune disease, experimental allergic encephalomyelitis.

Animals↗

PubMed2000

Factors affecting hepatocyte viability and CYPIA1 activity during encapsulation.

Hepatocytes encapsulated in alginate-poly-1-lysine-alginate (APA) are used in transplantation studies and in bioartificial liver support systems. Loss of cell viability in the process of APA encapsulation is usually 20-30% while the effect on cytochrome CYP450 activity is rarely reported. This work investigates the negative influences on hepatocyte viability and CYPIA1 activity during APA encapsulation, and reports methods to alleviate these influences by incorporating certain reagents into the encapsulation solution. The results show that loss of hepatocyte viability and CYPIA1 activity was caused almost entirely by extracellular calcium toxicity rather than by mechanical damage (p < 0.05). Use of 10 mM instead of 100 mM calcium chloride (CaCl2) in the encapsulation process improved CYPIA1 activity (p < 0.05), but did not improve hepatocyte viability (p > 0.05) or result in satisfactory microcapsules. Hepatocyte viability was 25% higher (p < 0.05) in CaCl2 than in calcium lactate (CaLa) when the cells were gelled by contact with these calcium solutions at room temperature (RT). Hepatocyte viability showed little improvement by processing at 4 degrees C than at RT in CaCl2 (p > 0.05) but was 23% higher at 4 degrees C than at RT in CaLa (p < 0.05). Calcium used in the process of encapsulation caused cell necrosis rather than apoptosis. Addition of Dulbecco's modified Eagle's medium (containing 10% foetal bovine serum) or 20 mM fructose to the calcium solution did not improve cell survival. However, nifedipine at a final concentration of 25 mM modestly improved hepatocyte survival in solution containing 100 mM CaCl2 (p = 0.003). Glutathione and taurine in certain concentrations showed protective effects against loss of CYPIA1 activity (p < 0.05 and <0.01 respectively). In conclusion, to optimise the use of calcium during the process of encapsulation, CaCl2 is preferred to CaLa and inclusion of nifedipine, glutathione or taurine in 100 mM CaCl2 solution is recommended.

Alginates↗

PubMed2000

Fatal methadone poisoning in children: Maryland 1992-1996.

The prevention of accidental poisoning by prescribed an over-the-counter medication has been successfully addressed by the use of child-resistant containers. Nevertheless. accidental methadone poisoning in children remains a problem. The Office of the Chief Medical Examiner for the State of Maryland has investigated four deaths in children due to methadone poisoning over a 4-year period. Three of the cases occurred within a 3-month period. Two victims accidentally ingested methadone within 3 days of each other. The authors address the continuing danger of methadone poisoning to children and identify factors contributing to this problem.

Accidents, Home↗

PubMed2000

Both locus control region and proximal regulatory elements direct the developmental regulation of beta-globin gene cluster.

Using ligation-mediated PCR and in vivo footprinting methods to study the status of DNA-protein interaction at hypersensitive site 2 of locus control region and beta(maj) promoter of erythroid cells of fetal liver and adult bone marrow, we found that during different developmental periods, the status of DNA-protein interaction at both hypersensitive site 2 and beta(maj) promoter changed significantly, and indicated that locus control region might function through a looping mechanism to regulate the expression of downstream genes, and that distal regulatory elements (locus control region, hypersensitive sites) as well as proximal regulatory elements (promoter, enhancer) of beta-globin gene cluster participate in the regulation of developmental specificity.

Animals↗

PubMed2000

[Detection of Prader-Willi syndrome by methylation-specific PCR].

OBJECTIVE: Using a faster and more efficient method to diagnose Prader-Willi syndrome(PWS). METHODS: Differential DNA methylation at several sites in the PWS critical region where the maternal homologue is unmethylated and transcriptionally active. Methylation-specific PCR(MSPCR) is based on sodium bisulfite treatment of DNA, which converts unmethylated but not methylated cytosine residues to uracil, and PCR primers specific for the maternal and the paternal allele. Methylation analysis by Southern blot is made to validate the MSPCR result. RESULTS: Bisulfite-modified DNA from PWS patients amplified only with methylated allele-specific primer pair showed only maternal 174bp PCR product. MSPCR and PW71B methylation studies showed an abnormal pattern consistent with the clinical diagnosis of PWS. Untreated DNA does not produce a PCR product. CONCLUSION: MSPCR can be used to detect all presently testable causes of PWS (deletion, uniparental disomy, and imprinting mutation) in an efficient first step for stepwise diagnostic testing, compared with either Southern blot analysis which is more timeconsuming, or fluorescence in situ hybridization(FISH) which can not detect uniparental disomy or imprinting mutation.

Adolescent↗

PubMed2000

Genetic modeling of human urinary bladder carcinogenesis.

We developed a model of human urinary bladder cancer progression from in situ precursor lesions to invasive carcinoma using whole organ histologic and genetic mapping. The model represents a high-density and detailed analysis regarding allelic losses on chromosomes 4, 8, 9, 11, and 17 as revealed by testing of 234 samples obtained from five cystectomy specimens. The samples corresponded to microscopically identified intraurothelial precursor conditions ranging from dysplasia to carcinoma in situ and invasive cancer. The initial analysis of paired normal and tumor DNA samples disclosed allelic losses in 72 of 225 tested hypervariable DNA markers. Subsequent use of these markers on all mucosal samples revealed that 47 had alterations with a statistically significant relation to urothelial neoplasia. The allelic losses clustered in 33 distinct chromosomal regions, indicating the location of putative tumor suppressor genes involved in the development and progression of urinary bladder cancer. Some of the markers with statistically significant allelic losses mapped to the regions containing well-characterized tumor suppressor genes but many were located in previously unknown loci. The majority of statistically significant allelic losses (70%) occurred early in low-grade intraurothelial dysplasia, and some of them involved adjacent areas of morphologically normal mucosa preceding the development of microscopically recognizable precursor lesions. The remaining 30% of markers developed allelic losses in the later phases of urothelial neoplasia, implicating their involvement in progression to invasive disease. Markers exhibiting allelic losses in early phases of urothelial neoplasia could be used for detection of occult preclinical or even premicroscopic phases of urinary bladder cancer, whereas markers that showed allelic losses in the later phases of the process could serve as indicators of progression to invasive disease. The approach used in this study facilitates genome-wide modeling of cancer progression and provides important chromosomal landmarks for more specific studies of multistep urinary bladder carcinogenesis.

Aged↗

PubMed2000

Expression of ERCC1 antisense RNA abrogates gemicitabine-mediated cytotoxic synergism with cisplatin in human colon tumor cells defective in mismatch repair but proficient in nucleotide excision repair.

Gemcitabine, or 2',2'-difluorodeoxycytidine (dFdC) is a new anticancer agent with significant activity against a broad spectrum of tumors either as a single agent or in combination with other active anticancer drugs. Studies in vitro and in vivo have demonstrated that dFdC produces cytotoxic synergism with cisplatin, or cis-diamminedi-choloroplatinum(II) (CDDP); however, the mechanism by which the synergism occurs has not been elucidated. We proposed that the nucleotide excision repair (NER) process, which is responsible for the cellular removal of CDDP-DNA adducts, may be a target for the mechanism of the cytotoxic synergism of dFdC and CDDP. Because the mismatch repair (MMR) pathway is involved in mediating CDDP cytotoxicity, making determination of the role of the NER in the cytotoxic synergism more complicated, and because tumors are often defective in MMR, we selected an NER-proficient, MMR-deficient, CP2.0 human colon carcinoma cell line as a model for this study. By an in vitro repair synthesis assay, we found that dFdC triphosphate (dFdCTP), the active metabolite of dFdC, inhibited the incorporation of [alpha-32P]dATP as well as the incorporation of [alpha-32P]dCTP, suggesting that the repair inhibition by dFdCTP does not result simply from competition for the incorporation site but rather is also due to prevention of chain elongation during the DNA resynthesis process. To determine whether the repair inhibition contributes to the cytotoxic synergism, we examined the effect of the constitutive expression of ERCC1 antisense RNA on the interaction of dFdC and CDDP. CP2.0 cells were transfected with pERCC1/AS, an ERCC1 antisense expression vector; eight hygromycine-resistant clones expressing various levels of the antisense RNA were selected for quantification of and correlation between the repair activity and cytotoxic synergism. The results show that stable expression of ERCC1 antisense RNA down-regulated the level of mRNA and repair activity; the down-regulation of the repair activity significantly correlated with the reduction of the cytotoxic synergism of the two agents. These data provide direct evidence to support the hypothesis that inhibition of the repair of CDDP-induced DNA lesions plays a critical role in dFdC-mediated cytotoxic synergism with CDDP in MMR-deficient tumor cells.

Antineoplastic Agents↗