Biomedical subjects

D Fink

Publications and source records attributed to D Fink.

At least 37 records · Page 2Linked to original sources

PubMed2000

Prognostic significance of CA 125 and TPS levels after 3 chemotherapy courses in ovarian cancer patients.

OBJECTIVE: To evaluate the prognostic significance of and predictive value for survival of CA 125 and TPS levels after three chemotherapy courses in ovarian cancer patients. METHODS: We analyzed in a prospective multicenter study the 1- and 2-year overall survival (OS) in ovarian carcinoma patients. The prognostic significance of CA 125 and TPS levels above the discrimination value (25 kU/L and 100 U/L, respectively) was examined by univariate and multivariate analyses. RESULTS: Of the 213 cases included, 64 patients were staged as FIGO I + II and 149 patients were staged as FIGO III + IV. Tumor marker levels in stage I + II were not correlated with survival. However, stage III and IV patients with elevated levels of CA 125 or TPS after three chemotherapy courses had a worse 2-year OS (69% vs 26%, P < 0.0001 and 57% vs 20%, P < 0.0001, respectively) than patients with normal levels of the markers. In univariate analysis the result of operation (staging laparatomy and partial debulking) and advanced FIGO stage (IV) were also adverse prognostic factors. Independent factors predictive of low 2-year OS by multivariate analysis were staging laparotomy, TPS elevated, and CA 125 elevated. The only factors predictive of low 1-year OS were TPS elevated and staging laparotomy. CONCLUSIONS: Ovarian cancer patients with elevated CA 125 levels after three chemotherapy courses have a poor prognosis. However, the prognostic accuracy can be significantly increased by the parallel determination of serum TPS.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2000

Transcriptional analysis of the gene for glutamine synthetase II and two upstream genes in Streptomyces coelicolor A3(2).

The glutamine synthetase II (GSII, encoded by glnII) activity detectable in crude extracts from Streptomyces coelicolor is low compared to the activity of glutamine synthetase I (GSI, encoded by glnA) and to that of GSII from S. viridochromogenes. We have identified and sequenced a 3.9-kb BglII-BamHI fragment carrying the glutamine synthetase II gene (glnII) from S. coelicolor. Besides glnII, this region contains four ORFs (orf1-orf4). While homologues of orf1 and orf2 were also found in the glnII region of the S. viridochromogenes chromosome, this was not the case for orf3 and orf4, which encode a putative hydrolase and a transcriptional regulator (Ptr) of the MarR family, respectively. High-resolution S1 nuclease mapping showed that the S. coelicolor glnII gene is expressed from two overlapping promoters. The first comprises a vegetative promoter sequence and the second contains sequence elements that are recognized by Esigma31. Similar promoter structures were found upstream of the S. viridochromogenes glnII gene. The involvement of ptr in glnII regulation was studied by gel retardation assays. Recombinant Ptr interacted with the upstream region of ptr, but not with the promoter region of glnII. A ptr gene replacement mutant (S. coelicolor IP) was also constructed. RT-PCR analysis of RNA from wild-type S. coelicolor and the IP mutant demonstrated that expression of orf3 depends on Ptr. Thus, the difference in gene organization between S. coelicolor and S. viridochromogenes is not responsible for the difference in GSII activity.

Bacterial Proteins↗

PubMed2000

Accelerator mass spectrometry analyses of environmental radionuclides: sensitivity, precision and standardisation

Accelerator Mass Spectrometry (AMS) is the analytical technique of choice for the detection of long-lived radionuclides which cannot be practically analysed with decay counting or conventional mass spectrometry. AMS allows an isotopic sensitivity as low as one part in 10(15) for 14C (5.73 ka), 10Be (1.6 Ma), 26Al (720 ka), 36Cl (301 ka), 41Ca (104 ka), 129I (16 Ma) and other long-lived radionuclides occurring in nature at ultra-trace levels. These radionuclides can be used as tracers and chronometers in many disciplines: geology, archaeology, astrophysics, biomedicine and materials science. Low-level decay counting techniques have been developed in the last 40-50 years to detect the concentration of cosmogenic, radiogenic and anthropogenic radionuclides in a variety of specimens. Radioactivity measurements for long-lived radionuclides are made difficult by low counting rates and in some cases the need for complicated radiochemistry procedures and efficient detectors of soft beta-particles and low energy x-rays. The sensitivity of AMS is unaffected by the half-life of the isotope being measured, since the atoms not the radiations that result from their decay, are counted directly. Hence, the efficiency of AMS in the detection of long-lived radionuclides is 10(6)-10(9) times higher than decay counting and the size of the sample required for analysis is reduced accordingly. For example, 14C is being analysed in samples containing as little as 20 microg carbon. There is also a world-wide effort to use AMS for the analysis of rare nuclides of heavy mass, such as actinides, with important applications in safeguards and nuclear waste disposal. Finally, AMS microprobes are being developed for the in-situ analysis of stable isotopes in geological samples, semiconductors and other materials. Unfortunately, the use of AMS is limited by the expensive accelerator technology required, but there are several attempts to develop compact AMS spectrometers at low (< or = 0.5 MV) terminal voltages. Recent advances in AMS will be reviewed with highlights from the scientific programs at Lucas Heights and other AMS centres.

Journal Article↗

PubMed2000

Isolation and expression in Escherichia coli of cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum.

In medium supplemented with chondroitin sulfate, Flavobacterium heparinum synthesizes and exports two chondroitinases, chondroitinase AC (chondroitin AC lyase; EC 4.2.2.5) and chondroitinase B (chondroitin B lyase; no EC number), into its periplasmic space. Chondroitinase AC preferentially depolymerizes chondroitin sulfates A and C, whereas chondroitinase B degrades only dermatan sulfate (chondroitin sulfate B). The genes coding for both enzymes were isolated from F. heparinum and designated cslA (chondroitinase AC) and cslB (chondroitinase B). They were found to be separated by 5.5 kb on the chromosome of F. heparinum, transcribed in the same orientation, but not linked to any of the heparinase genes. In addition, the synthesis of both enzymes appeared to be coregulated. The cslA and cslB DNA sequences revealed open reading frames of 2,103 and 1,521 bp coding for peptides of 700 and 506 amino acid residues, respectively. Chondroitinase AC has a signal sequence of 22 residues, while chondroitinase B is composed of 25 residues. The mature forms of chondroitinases AC and B are comprised of 678 and 481 amino acid residues and have calculated molecular masses of 77,169 and 53,563 Da, respectively. Truncated cslA and cslB genes have been used to produce active, mature chondroitinases in the cytoplasm of Escherichia coli. Partially purified recombinant chondroitinases AC and B exhibit specific activities similar to those of chondroitinases AC and B from F. heparinum.

Blotting, Southern↗

PubMed2000

[Neurofibromatosis of the breast in a patient with Morbus von Recklinghausen].

Neurofibromatosis is a rare autosomal dominant disorder with several subtypes. Common is the appearance of specific skin alterations. Neurofibromas occurring in the breast are very rare, and in such cases they are most common in the areolar area. A case of a 46-year-old woman with von Recklinghausen's disease of the breast is reported, and the literature will be discussed.

Breast Neoplasms↗

PubMed2000

Analysis of MLH1 and MSH2 expression in ovarian cancer before and after platinum drug-based chemotherapy.

Preclinical studies have demonstrated a relationship between DNA mismatch repair (MMR) status and sensitivity to cisplatin and carboplatin. MMR-deficient cells are resistant to both drugs, and selection for cisplatin resistance in vitro is sometimes accompanied by loss of MMR protein expression. We used immunohistochemical staining techniques to investigate hMLH1 and hMSH2 expression in paired ovarian tumor sections from 54 ovarian cancer patients before and after platinum-based therapy. We sought associations between hMLH1 and hMSH2 protein expression and clinical parameters known to be of prognostic significance as well as response to treatment and overall survival. hMLH1 and hMSH2 staining decreased significantly after platinum-based therapy. The percent of malignant cells that stained positive correlated with the intensity of nuclear staining for both proteins; staining for hMLH1 correlated well with staining for hMSH2. Unexpectedly, expression of nuclear hMLH1 correlated negatively with response to treatment. Expression of nuclear hMLH1 and hMSH2 was positively correlated with pretreatment CA125 level, and expression of nuclear hMSH2 was positively correlated with change in CA125 level after treatment. Tumor stage was associated with expression of nuclear hMSH2, and tumor histological subtype was associated with both hMLH1 and hMSH2 staining. No association was found between expression of either protein and overall survival. These results indicate that the tumor is biologically altered after chemotherapy consistent with treatment-induced selection for cells expressing lower hMLH1 and hMSH2 levels. However, immunohistochemical staining for either hMLH1 or hMSH2 was not highly predictive of drug sensitivity as measured by response or survival.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Primary carcinoma of the fallopian tube. A report of 19 cases with literature review.

Primary carcinoma of the fallopian tube is the rarest cancer of the female genital tract with an incidence of 0.5% of all gynecologic tumors. Since the first report in 1847 about 1,500 cases have been published. Due to similarity of the clinical presentation the staging and therapeutic management have been adapted to that of ovarian cancer. We retrospectively evaluated all the 19 patients who had been diagnosed with primary carcinoma of the fallopian tube at the Department of Obstetrics and Gynecology of the University of Zurich between 1977 and 1998. All lesions were staged according to the rules of FIGO adopted in 1991. At the time of diagnosis the median age was 62 (46-87) years. Twelve (63%) women revealed FIGO stage III-IV, whereas four (21%) and three (16%) patients were diagnosed in stage I and stage II, respectively. Eight (42%) women were nullipara. Histology showed serous-papillary carcinoma, in ten (53%) cases. The 5-year survival rate was 22% for all FIGO stages and 80% for stage I. None of the patients with stage III and IV survived 5 years. Ovarian cancer and primary carcinoma of the fallopian tube are similar in many aspects. Both carcinomas have a similar age distribution, show an increase among nulliparous women, are often of serous papillary histology, have a poor prognosis with stage and residual tumor size as important prognostic factors, and respond initially well to platinum-based chemotherapy. Nevertheless, there appears to be a difference between the two diseases: primary carcinoma of the fallopian tube is more often diagnosed in an earlier stage. This many be due to lower abdominal pain resulting from tubal dilatation and to abnormal bloody-watery discharge.

Adult↗

PubMed1999

Decreased cisplatin damage-dependent DNA synthesis in cellular extracts of mismatch repair deficient cells.

The proficiency of both nucleotide excision repair (NER) and DNA mismatch repair (MMR) influences cellular sensitivity to cisplatin (cis-diamminedichloroplatinum). To gain further insight into how MMR may influence platinum drug sensitivity, the effect of loss of MMR on repair synthesis was measured in vitro by a commonly used method that relies on whole-cell extracts to drive [alpha-32P]dATP incorporation into cisplatin-damaged plasmid DNA. Extracts evaluated include those from cells with or without functional hMLH1 (HCT116+ch2 versus HCT116+ch3, respectively) and hMSH2 (HEC59 versus HEC59+ch2, respectively). Loss of MMR in the HCT116 system was associated with a 2.8-fold reduction in cisplatin damage-specific DNA synthesis, whereas it was associated with a 3.0-fold reduction in the HEC59 system, suggesting that a decrease in the ability to repair cisplatin-damaged DNA accompanies loss of MMR. An in vitro DNA excision assay that utilized a substrate containing a site-specific cisplatin adduct was performed. Using this highly NER-specific assay, no significant difference was apparent between the extracts derived from NER-proficient versus -deficient cells. These and other data lead us to suggest that the increase in apparent repair synthesis in platinum-damaged plasmids by extracts from MMR-proficient versus -deficient cellular extracts may reflect a distinct and possibly adverse DNA synthetic process rather than productive NER.

Base Pair Mismatch↗

PubMed1999

Photodynamic therapy for the hypercalcemic type of the small cell carcinoma of the ovary in a mouse xenograft model.

OBJECTIVE: The hypercalcemic type of the small cell carcinoma of the ovary (HTSCCO) is a rapidly fatal ovarian tumor in young women. Photodynamic therapy (PDT) induces selective necrosis to malignant tissues. The aim of this study was to determine the sensitivity of the HTSCCO to PDT in a mouse xenograft model. METHODS: Tumors were obtained from a patient with a HTSCCO and were transplanted into nude mice. Following photosensitization with m-THPC, either superficial or interstitial laser light was administered to the tumors. Necroses were measured by morphometry. Serum calcium levels were determined prior to and after PDT. RESULTS: Superficial irradiation of m-THPC sensitized tumors showed over three times more necrosis than control tumors (P = 0.037). Interstitially irradiated tumors showed over seven times more necrosis than control tumors (P = 0.0012). All animals showed a highly significant hypercalcemia prior to PDT (P < 0.0001). PDT induced a significant decrease in serum calcium levels (P = 0.0297). CONCLUSION: This study suggests that PDT may be of therapeutic value for the HTSCCO.

Animals↗

PubMed1999

Heterologous upregulation of nerve growth factor-TrkA receptors in PC12 cells by pituitary adenylate cyclase-activating polypeptide (PACAP).

The capacity for the neurotrophic factor PACAP38 to regulate expression of nerve growth factor (NGF)-trkA receptors in PC12 cells has been examined. Treatment of PC12 cells with 5 nM PACAP38 for 48 h elicited a 2.5-fold increase in 125I-NGF binding sites. FACS and Western analysis of trkA receptor protein indicate an abundance of receptors. The PACAP38-selective antagonist PACAP 6-38 blocked trkA receptor upregulation elicited by PACAP38. The expression of epidermal growth factor receptors was not affected by PACAP38 suggesting that upregulation of trkA represents a selective effect of this neurotrophic peptide. Similarly, expression of the pan-neurotrophin binding receptor p75 was not altered by PACAP38 treatment. In addition to effects on trkA observed in wild-type PC12 cells, PACAP38 stimulated an increase in the level of expressed human trkA receptors stably transfected into PC12 cells. PACAP38 provoked an increase in basal and NGF-stimulated phosphorylation of trkA. Enhanced phosphorylation of trkA was detected as early as 6 h following addition of PACAP38 and was maximal at 48 h. Increased incorporation of phosphate occurs on both serine and tyrosine residues of trkA. These results suggest that PACAP38 is able to promote upregulation of trkA receptors, an event associated with elevated serine/tyrosine phosphorylation of trkA.

Animals↗

PubMed1999

A phase II/pharmacokinetic trial of high-dose progesterone in combination with paclitaxel.

PURPOSE: The purpose of this study was to investigate the effect of high-dose progesterone, an inhibitor of P glycoprotein, on the pharmacokinetics and toxicity of paclitaxel. PATIENTS AND METHODS: A total of 29 patients with various tumors were treated with single-agent paclitaxel (125 mg/m2 administered over 3 h once every 3 weeks) until progression of disease, at which point high-dose progesterone (3 g administered i.v. over 24 h) was added to the paclitaxel treatment program in 20 patients (13 women, 7 men). Pharmacokinetic studies of paclitaxel administered alone and with progesterone were performed in eight patients. RESULTS: The pharmacokinetic parameters of paclitaxel were highly variable. High-dose progesterone increased the peak plasma levels (3.00 +/- 0.94 vs. 4.15 +/- 1.63 microM; P = 0.029; mean +/- SD) and the area under the curve (AUC; 14.3 +/- 4.75 vs. 17.3 +/- 5.59 microM x h; P = 0.006) of paclitaxel. The absolute neutrophil and platelet nadir counts did not differ significantly between the paclitaxel and the combined treatment cycles. Three of the 20 patients documented to have progressive disease on paclitaxel alone had partial responses when high-dose progesterone was added to the paclitaxel regimen. CONCLUSION: Progesterone had a statistically significant impact on the pharmacokinetics of paclitaxel. The addition of high-dose progesterone to paclitaxel is feasible, but the small number of patients prevents conclusions being drawn about the clinical efficacy of combined progesterone and paclitaxel.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Mismatch repair proficiency and in vitro response to 5-fluorouracil.

BACKGROUND & AIMS: The DNA mismatch repair (MMR) system recognizes certain DNA adducts caused by alkylation damage in addition to its role in recognizing and directing repair of interstrand nucleotide mismatches and slippage mistakes at microsatellite sequences. Because defects in the MMR system can confer tolerance to acquired DNA damage and, by inference, the toxic effects of certain chemotherapeutic agents, we investigated the effect of 5-fluorouracil (5-FU) on colon cancer cell lines. METHODS: We determined growth selection by cell enrichment assay and cloning efficiency after treatment with 5 micromol/L 5-FU, assayed nucleic 3H-5-FU incorporation, and analyzed the cell cycle by flow cytometry. RESULTS: 5-FU treatment provided a growth advantage for MMR-deficient cell lines, indicating a relative degree of tolerance to 5-FU by the MMR-deficient cell lines. Enhanced survival was statistically significant after 5 days of growth, and a 28-fold reduction in survival was noted in the MMR-proficient cells by clonagenic assays after 10 days of growth. Differences in nucleotide uptake of 5-FU did not account for the observed growth differences, and specific cell cycle checkpoint arrest was not detected. CONCLUSIONS: Intact DNA MMR seems to recognize 5-FU incorporated into DNA but may do so in a different manner than other types of alkylation damage. Defective DNA MMR might be one mechanism for tumor resistance to 5-FU.

Animals↗

PubMed1999

Induction of JNK and c-Abl signalling by cisplatin and oxaliplatin in mismatch repair-proficient and -deficient cells.

Loss of DNA mismatch repair has been observed in a variety of human cancers. Recent studies have shown that loss of DNA mismatch repair results in resistance to cisplatin but not oxaliplatin, suggesting that the mismatch repair proteins serve as a detector for cisplatin but not oxaliplatin adducts. To identify the signal transduction pathways with which the detector communicates, we investigated the effect of loss of DNA mismatch repair on activation of known damage-responsive pathways, and recently reported that cisplatin differentially activates c-Jun NH2-terminal kinase (JNK) and c-Abl in repair-proficient vs.-deficient cells. In the current study, we directly compared differential activation of these pathways by cisplatin vs. oxaliplatin. The results confirm that cisplatin activates JNK kinase 5.7 +/- 1.5 (s.d.)-fold more efficiently in DNA mismatch repair-proficient than repair-deficient cells, and that the c-Abl response to cisplatin is completely absent in DNA mismatch repair-deficient cells. In contrast, there was no detectable activation of the JNK or c-Abl kinases in DNA mismatch repair-proficient or -deficient cells exposed to oxaliplatin. The present study demonstrates that, despite the similarity of the adducts produced by cisplatin and oxaliplatin, they appear to be recognized by different detectors. The DNA mismatch repair system plays an important part in the recognition of cisplatin adducts, and activation of both the JNK and c-Abl kinases in response to cisplatin damage is dependent on the detector function of the DNA mismatch repair proteins. In contrast, this detector does not respond to oxaliplatin adducts.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Loss of DNA mismatch repair facilitates reactivation of a reporter plasmid damaged by cisplatin.

In addition to recognizing and repairing mismatched bases in DNA, the mismatch repair (MMR) system also detects cisplatin DNA adducts and loss of MMR results in resistance to cisplatin. A comparison was made of the ability of MMR-proficient and -deficient cells to remove cisplatin adducts from their genome and to reactivate a transiently transfected plasmid that had previously been inactivated by cisplatin to express the firefly luciferase enzyme. MMR deficiency due to loss of hMLH1 function did not change the extent of platinum (Pt) accumulation or kinetics of removal from total cellular DNA. However, MMR-deficient cells, lacking either hMLH1 or hMSH2, generated twofold more luciferase activity from a cisplatin-damaged reporter plasmid than their MMR-proficient counterparts. Thus, detection of the cisplatin adducts by the MMR system reduced the efficiency of reactivation of the damaged luciferase gene compared to cells lacking this detector. The twofold reduction in reactivation efficiency was of the same order of magnitude as the difference in cisplatin sensitivity between the MMR-proficient and -deficient cells. We conclude that although MMR-proficient and -deficient cells remove Pt from their genome at equal rates, the loss of a functional MMR system facilitates the reactivation of a cisplatin-damaged reporter gene.

Adenocarcinoma↗

PubMed1999

Nitrogen metabolism in Streptomyces coelicolor A3(2): modification of glutamine synthetase I by an adenylyltransferase.

An internal adenylyltransferase gene (glnE) fragment from Streptomyces coelicolor was amplified using heterologous PCR primers derived from consensus motifs. The sequence had significant similarity to bacterial glnE genes, and included a motif typical of the C-terminal adenylyltransferase domain of glnE. glnE from S. coelicolor lies on the Asel-C fragment of the chromosome and is localized near glnA (encoding glutamine synthetase I, GSI) and glnII (encoding GSII). To analyse the function of glnE in S. coelicolor, glnE (S. coelicolor E4) and glnA (S. coelicolor HT107) gene replacement mutants were constructed. The GSI activity of the glnE mutant was not down-regulated after an ammonium shock. However, the GSI activity of the wild-type cells decreased to 60% of the original activity. The glnA mutant is not glutamine auxotrophic, but in the gamma-glutamyltransferase assay no GSI activity was detected in unshifted and shifted HT107 cells. By snake venom phosphodiesterase treatment the GSI activity in the wild-type can be reconstituted, whereas no alteration is observed in the E4 mutant. Additionally, the loss of short-term GSI regulation in the E4 mutant was accompanied by an increased glutamine:glutamate ratio.

Amino Acid Sequence↗

PubMed1999

The role of the frequency-volume chart in the differential diagnostic of female urinary incontinence.

OBJECTIVE: To investigate the differential diagnostic capacity of the frequency-volume chart in 132 women with urinary incontinence. MATERIAL AND METHOD: For each patient, the readings of two 24-h frequency-volume charts were compared to the urodynamic diagnosis which was used as the gold standard in 73 genuine stress incontinent women, in 23 urge incontinent women, and in 36 women with mixed incontinence. RESULTS: The total voided volume, the mean voided volume, the largest single voided volume, and the smallest single voided volume were statistically differentiating single parameters among the three groups (p<0.05). Applying logistic regression to the two well-defined groups of patients, the one with urge incontinence and the one with genuine stress incontinence, the frequency of micturition during nighttime revealed the best discriminatory power for these conditions. CONCLUSION: This study shows that in the frequency-volume chart micturition during nighttime discriminates best between urge and stress incontinence.

Adult↗

PubMed1998

Enhanced tumor cell killing in the presence of ganciclovir by herpes simplex virus type 1 vector-directed coexpression of human tumor necrosis factor-alpha and herpes simplex virus thymidine kinase.

Past studies have documented the promise of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) suicide gene therapy as a potential antitumor treatment. HSV-TK converts the pro-drug ganciclovir (GCV) into a toxic nucleotide analogue, the incorporation of which into cellular DNA blocks cell proliferation. In this report, we have examined the hypothesis that the effectiveness of HSV-TK suicide gene therapy can be enhanced by coexpression of the antitumor cytokine human tumor necrosis factor-alpha (TNF-alpha) from the same replication-defective HSV-1 vector. In vitro testing demonstrated that TNF-alpha expression from this vector potentiated the killing of both TNF-alpha-sensitive L929 tumor cells and TNF-alpha-resistant U-87 MG cells in the presence of GCV. Furthermore, treatment of established intradermal L929 tumors in vivo with the TNF-alpha/TK vector and GCV resulted in prolonged animal survival compared with treatment with parental HSV-TK vector in the presence or absence of GCV. Treatment of intracerebral U-87 MG tumors showed a clear benefit of TK therapy, but a significant further increase in survival using the TNF-alpha vector could not be demonstrated. We found that potentiation of cell killing in vitro required intracellular TNF-alpha because purified protein added to the culture medium of cells infected with HSV-TK vector failed to have the same effect. Accordingly, potentiation in vivo should depend on efficient infection, but immunohistochemical analysis indicated that virus administration by U-87 MG intratumoral injection was inadequate, resulting in an estimated <1% infection of all tumor cells. Moreover, the majority of infected tumor cells were localized at the tumor margin. Together, these results suggest that TNF-enhanced tk gene therapy should provide a useful treatment for TNF-alpha-sensitive tumors and perhaps also for TNT-alpha-resistant tumors if vector delivery can be improved to increase the percentage of transduced tumor cells.

Animals↗

PubMed1998

Enrichment for DNA mismatch repair-deficient cells during treatment with cisplatin.

In addition to playing a role in tumorigenesis, loss of DNA mismatch repair results in low-level intrinsic resistance to cisplatin and carboplatin. We used a mismatch repair-deficient (clone B) and -proficient (clone B/rev) pair of Chinese hamster ovary sublines to determine the ability of cisplatin to enrich for repair-deficient cells during growth in vitro and in vivo. Clone B cells were 1.8-fold resistant to cisplatin as measured by a clonogenic assay. These cells were molecularly engineered to express constitutively the green fluorescent protein, and changes in the fraction of these repair-deficient cells were monitored by flow cytometric analysis. A single 1-hr exposure to cisplatin at an IC50 concentration enriched populations initially containing either 5 or 10% clone B cells by 81 and 75%, respectively, when measured at 5 days. Enrichment increased as a function of drug concentration to 158 and 169%, respectively, following an IC90 exposure. When grown as a xenograft, a single LD10 dose of cisplatin enriched the tumors by 48% from 4.6 to 6.8% repair-deficient cells (p = 0.04). To determine whether similar enrichment occurs during the treatment of human ovarian cancer patients, paired tumor samples were obtained from 38 patients before and after treatment with a minimum of 3 cycles of platinum drug-based primary chemotherapy and analyzed immunohistochemically for changes in the fraction of tumor cells expressing hMHL1. Following treatment there was a reduction in hMLH1 staining in 66% of the cases (p = 0.0005). Our results demonstrate that, despite the fact that loss of mismatch repair yields only modest levels of cisplatin resistance, even a single exposure to cisplatin produces quite a marked enrichment for repair-deficient cells in vitro and in vivo. Our results are consistent with the concept that treatment with cisplatin or carboplatin selects for preexisting mismatch repair-deficient cells, and that this contributes to the frequent development of clinical resistance.

Adaptor Proteins, Signal Transducing↗