Mismatch Repair Deficient Cell Lines Research Articles

DNA mismatch repair protects the genome from oxygen-induced replicative mutagenesis.

DNA mismatch repair (MMR) corrects mismatched DNA bases arising from multiple sources including polymerase errors and base damage. By detecting spontaneous mutagenesis using whole genome sequencing of cultured MMR deficient human cell lines, we show that a primary role of MMR is the repair of oxygen-induced mismatches. We found an approximately twofold higher mutation rate in MSH6 deficient DLD-1 cells or MHL1 deficient HCT116 cells exposed to atmospheric conditions as opposed to mild hypoxia, which correlated with oxidant levels measured using electron paramagnetic resonance spectroscopy. The oxygen-induced mutations were dominated by T to C base substitutions and single T deletions found primarily on the lagging strand. A broad sequence context preference, dependence on replication timing and a lack of transcriptional strand bias further suggested that oxygen-induced mutations arise from polymerase errors rather than oxidative base damage. We defined separate low and high oxygen-specific MMR deficiency mutation signatures common to the two cell lines and showed that the effect of oxygen is observable in MMR deficient cancer genomes, where it best correlates with the contribution of mutation signature SBS21. Our results imply that MMR corrects oxygen-induced genomic mismatches introduced by a replicative process in proliferating cells.

Two main mutational processes operate in the absence of DNA mismatch repair

The analysis of tumour genome sequences has demonstrated high rates of base substitution mutagenesis upon the inactivation of DNA mismatch repair (MMR), and the resulting somatic mutations in MMR deficient tumours appear to significantly enhance the response to immune therapy. A handful of different algorithmically derived base substitution mutation signatures have been attributed to MMR deficiency in tumour somatic mutation datasets. In contrast, mutation data obtained from whole genome sequences of isogenic wild type and MMR deficient cell lines in this study, as well as from published sources, show a more uniform experimental mutation spectrum of MMR deficiency. In order to resolve this discrepancy, we reanalysed mutation data from MMR deficient tumour whole exome and whole genome sequences. We derived two base substitution signatures using non-negative matrix factorisation, which together adequately describe mutagenesis in all tumour and cell line samples. The two new signatures broadly resemble COSMIC signatures 6 and 20, but perform better than existing COSMIC signatures at identifying MMR deficient tumours in mutation signature deconstruction. We show that the contribution of the two identified signatures, one of which is dominated by C to T mutations at CpG sites, is biased by the different sequence composition of the exome and the whole genome. We further show that the identity of the inactivated MMR gene, the tissue type, the mutational burden or the patient’s age does not influence the mutation spectrum, but that a tendency for a greater contribution by the CpG mutational process is observed in tumours as compared to cultured cells. Our analysis suggest that two separable mutational processes operate in the genomes of MMR deficient cells.

TGF-β signaling proteins and CYP24A1 may serve as surrogate markers for progesterone calcitriol treatment in ovarian and endometrial cancers of different histological types

Background: Strategies are needed to coordinately block drivers and induce suppressors of cancer to reduce incidence and improve outcomes for individuals with inherited or acquired risk. We previously reported the chemopreventive and therapeutic efficacy of the combination of progestin and calcitriol in transformed and malignant endometrioid endometrial cancer (EC) and in ovarian cancer models involving attenuated expression of TGF-β signaling proteins and progestin-mediated inhibition of calcitriol-induced CYP24A1 expression. This study aims to expand the applications for this combination to other subtypes of endometrial and ovarian cancers, including those with mutations in ARID1A or PIK3CA, DNA mismatch repair (MMR) deficiency or BRCA1 null status. Methods: Ovarian and EC cell lines of different histotypes were cultured with either progesterone, calcitriol, or the combination of progesterone and calcitriol for 3 or 5 days. The end points for this in vitro investigation included assessments of cell growth by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assays and the expression of TGF-β ligands, receptors, SMAD proteins and CYP24A1 by western blotting. Results: Treatment of ovarian clear cell carcinoma, endometrioid carcinoma, papillary serous adenocarcinoma, BRCA1 null, and DNA MMR deficient EC cell lines with progesterone alone or in combination with calcitriol inhibited cell growth and expression of TGF-β1, TGF-β2, TGF-Rβ1, TGF-βR2, pSMAD2/3 and CYP24A1. Expression of TGF-βR3, SMAD-4, progesterone receptor (PR) and vitamin-D receptor (VDR) was not altered in any cell line tested except, ES-2, where VDR expression was upregulated in response to treatment. Conclusions: These results suggest that progesterone alone and progesterone-calcitriol combination have broad application in both chemopreventive and therapeutic settings that merit further development in a wide variety of ovarian and ECs, including those derived from germline or somatic mechanisms. Moreover, our data suggest that TGF-β signaling proteins and CYP24A1 may be effective surrogate markers indicative of treatment response.

TGF-β signaling proteins and CYP24A1 may serve as surrogate markers for progesterone calcitriol treatment in ovarian and endometrial cancers of different histological types.

BackgroundStrategies are needed to coordinately block drivers and induce suppressors of cancer to reduce incidence and improve outcomes for individuals with inherited or acquired risk. We previously reported the chemopreventive and therapeutic efficacy of the combination of progestin and calcitriol in transformed and malignant endometrioid endometrial cancer (EC) and in ovarian cancer models involving attenuated expression of TGF-β signaling proteins and progestin-mediated inhibition of calcitriol-induced CYP24A1 expression. This study aims to expand the applications for this combination to other subtypes of endometrial and ovarian cancers, including those with mutations in ARID1A or PIK3CA, DNA mismatch repair (MMR) deficiency or BRCA1 null status.MethodsOvarian and EC cell lines of different histotypes were cultured with either progesterone, calcitriol, or the combination of progesterone and calcitriol for 3 or 5 days. The end points for this in vitro investigation included assessments of cell growth by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assays and the expression of TGF-β ligands, receptors, SMAD proteins and CYP24A1 by western blotting.ResultsTreatment of ovarian clear cell carcinoma, endometrioid carcinoma, papillary serous adenocarcinoma, BRCA1 null, and DNA MMR deficient EC cell lines with progesterone alone or in combination with calcitriol inhibited cell growth and expression of TGF-β1, TGF-β2, TGF-Rβ1, TGF-βR2, pSMAD2/3 and CYP24A1. Expression of TGF-βR3, SMAD-4, progesterone receptor (PR) and vitamin-D receptor (VDR) was not altered in any cell line tested except, ES-2, where VDR expression was upregulated in response to treatment.ConclusionsThese results suggest that progesterone alone and progesterone-calcitriol combination have broad application in both chemopreventive and therapeutic settings that merit further development in a wide variety of ovarian and ECs, including those derived from germline or somatic mechanisms. Moreover, our data suggest that TGF-β signaling proteins and CYP24A1 may be effective surrogate markers indicative of treatment response.

Cooperative recognition of T:T mismatch by echinomycin causes structural distortions in DNA duplex.

Small-molecule compounds that target mismatched base pairs in DNA offer a novel prospective for cancer diagnosis and therapy. The potent anticancer antibiotic echinomycin functions by intercalating into DNA at CpG sites. Surprisingly, we found that the drug strongly prefers to bind to consecutive CpG steps separated by a single T:T mismatch. The preference appears to result from enhanced cooperativity associated with the binding of the second echinomycin molecule. Crystallographic studies reveal that this preference originates from the staggered quinoxaline rings of the two neighboring antibiotic molecules that surround the T:T mismatch forming continuous stacking interactions within the duplex. These and other associated changes in DNA conformation allow the formation of a minor groove pocket for tight binding of the second echinomycin molecule. We also show that echinomycin displays enhanced cytotoxicity against mismatch repair-deficient cell lines, raising the possibility of repurposing the drug for detection and treatment of mismatch repair-deficient cancers.

Abstract 4387: The clonal composition of colorectal cancer cell lines is defined by the maintenance of specific genomic imbalances, not by ongoing chromosomal instability

Abstract Tumor type specific genomic imbalances are the defining feature of carcinomas. It remains elusive to which extent intratumor heterogeneity and the clonal composition of cancer cell populations influences the genetic makeup and the phenotype of tumors. Many studies aimed at understanding the idiosyncrasies of cancer rely on the analysis of cell lines derived from primary tumors. In an attempt to decipher patterns of chromosomal heterogeneity we analyzed the widely used colorectal cancer cell lines DLD-1, HCT116, H508, SW620, HT-29 and SW480 using FISH and gene expression profiling. First, we applied a four color probe set that targets the oncogenes EGFR, CCND1, CDX2 and TERC and enumerated 200 metaphase spread, which included chromosome counts, and 5,000 interphase cells. Not surprisingly, the mismatch repair deficient cell lines DLD-1 and HCT116 were the most stable, whereas the aneuploid cell lines displayed a higher degree of instability. These results prompted us to apply our recently developed multiplex interphase FISH approach that allows copy number analysis of 12 gene loci in individual cells to 11 single cell derived clones from both HT-29 and SW480. SW480 was considerably more instable than HT-29. The parental lines consisted of major clones, which were propagated in the single cell derived lines. These analyses were complemented by gene expression profiling of the single cell derived clones and the parental cell lines using the Nanostring technology which measures expression levels of 770 cancer-associated genes. The SW480 clones separated into two distinct clusters that reflect discrete ploidy levels that were also present in the parental line. We then explored whether mutations of the BRAF (HT-29) and KRAS (SW480) genes observed in the parental lines were maintained in the derived clones; this was the case in all instances. Finally, we reconstructed phylogenetic trees of tumor evolution using a specifically developed algorithm, termed FISHTrees. Our meticulous analysis of the clonal composition of these colorectal cancer models shows that, despite a certain degree of chromosomal instability, specific genomic imbalances are maintained. The results challenge the concept of ongoing chromosomal instability in cancer cell populations. This suggests a karyotype evolution that is driven by the necessity to arrive at a different plateau of chromosomal copy number as the driving force of carcinogenesis. Citation Format: Darawalee Wangsa, Madison Schiefer, Daniel Bronder, Hesed Padilla-Nash, Irianna Torres, Lidia Warner, Yue Hu, E Michael Gertz, Russell Schwartz, Alejandro A. Schäffer, Daniela Hirsch, Timo Gaiser, Rüdiger Meyer, Jordi Camps, Kerstin Heselmeyer-Haddad, Thomas Ried. The clonal composition of colorectal cancer cell lines is defined by the maintenance of specific genomic imbalances, not by ongoing chromosomal instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4387. doi:10.1158/1538-7445.AM2017-4387

A mononucleotide repeat in PRRT2 is an important, frequent target of mismatch repair deficiency in cancer.

The DNA mismatch repair (MMR) system corrects DNA replication mismatches thereby contributing to the maintenance of genomic stability. MMR deficiency has been observed in prostate cancer but its impact on the genomic landscape of these tumours is not known. In order to identify MMR associated mutations in prostate cancer we have performed whole genome sequencing of the MMR deficient PC346C prostate cancer cell line. We detected a total of 1196 mutations in PC346C which was 1.5-fold higher compared to a MMR proficient prostate cancer sample (G089). Of all different mutation classes, frameshifts in mononucleotide repeat (MNR) sequences were significantly enriched in the PC346C sample. As a result, a selection of genes with frameshift mutations in MNR was further assessed regarding its mutational status in a comprehensive panel of prostate, ovarian, endometrial and colorectal cancer cell lines. We identified PRRT2 and DAB2IP to be frequently mutated in MMR deficient cell lines, colorectal and endometrial cancer patient samples. Further characterization of PRRT2 revealed an important role of this gene in cancer biology. Both normal prostate cell lines and a colorectal cancer cell line showed increased proliferation, migration and invasion when expressing the mutated form of PRRT2 (ΔPRRT2). The wild-type PRRT2 (PRRT2wt) had an inhibitory effect in proliferation, consistent with the low expression level of PRRT2 in cancer versus normal prostate samples.

OP01 * MISMATCH REPAIR DEFICIENCY CONFERS INCREASED SENSITIVITY TO IONISING RADIATION (XR) IN GLIOBLASTOMA

INTRODUCTION: Combination chemo-radiotherapy, including the alkylating agent temozolomide (TMZ) is standard of care for patients with glioma. Increased TMZ use has lead to sustained interest in the mechanisms underlying acquired resistance. Amongst these, deficiency in mismatch repair (MMR) is best described, with TMZ treatment promoting loss of functional MMR proteins most frequently through point mutations in MSH2/6, which may then lead to a hyper-mutation phenotype. Since patients are increasingly exposed to TMZ as first-line treatment with subsequent radiotherapy we set out to investigate how MMR status may influence response to XR and/or radiosensitising agents. METHOD: Effects of MMR protein deficiency on response to XR in paired isogenic glioma cell lines; U251 (MMR proficient) and U251.TR3 (MMR deficient, TMZ-induced MSH6mut) lines determined by clonogenic assay and immunofluorescence to detect γH2AX foci. MMR proteins were down-regulated by siRNA transfection. MMR protein expression was determined by western blotting and MMR deficient cell lines were further characterised by sequencing. RESULTS: We have shown that U251.TR3 cells (MMR deficient) are more sensitive to radiation-induced cell death than MMR proficient U251 cells in clonogenic assays (surviving fraction at 2Gy 0.41 ± 0.03 and 0.71 ± 0.04 respectively, p < 0.001). This sensitisation is accompanied by an increase in gH2AX foci (28 ± 3 and 50 ± 6 foci per cell in U251 and U251.TR3 cells respectively). On-going work is investigating whether this effect is due to MMR deficiency per se or secondary mutations driven by a hyper-mutation phenotype. CONCLUSION: Detection of MMR proteins following TMZ exposure may serve as a tool to stratify patients for treatment.

Synergistic Cytotoxic Effect Of The PARP Inhibitor ABT888 and Busulfan In Acute Leukemia Cell Lines

The alkylator Busulfan (Bu) is used in conditioning regimens for acute leukemia prior to allogeneic stem cell transplant. Multiple DNA repair mechanisms including mismatch repair and base excision repair have been implicated in resistance to Bu. The enzyme PARP is central to base excision repair. We hypothesized that treatment of acute leukemia cell lines with both ABT888 (Veliparib), an inhibitor of PARP 1 and 2, and Bu would lead to synergistic cell kill and that this effect is maximal in mismatch repair deficient cells.Two mismatch repair proficient cell lines (K562 and HL60) and 2 mismatch repair deficient cell lines (NB4 and REH) were treated with ABT888 alone, Bu alone or a combination of both. In single drug experiments, doses of drug treatment ranged from 0-400mcg/ml. In combination experiments a fixed dose of ABT888 of 1.25mcg/ml was utilized with Bu doses varying from 0-200mcg/ml. This dose of ABT888 was chosen as it approximated to patient blood levels in clinical trials. After 24 hours of treatment, cells were washed and resuspended in fresh medium. Proliferation of cells was measured by standard 3H-thymidine uptake assay at 48 hours. Sigmoidal dose response curves and GI50 values were then calculated. In addition, cells were tested for apoptosis by flow cytometry using activated caspase 3 and annexin/ PI staining at 24 and 48 hours after treatment.All 4 cell lines were found to be resistant to single agent ABT888. Despite mismatch repair deficiency in REH cells, therapeutic doses of ABT888 did not cause significant decreases in proliferation. The effect of ABT888 was, as expected, much less evident in the mismatch repair proficient K562 cells. These cells were also relatively resistant to single agent Bu in vitro. The combination of Bu and ABT888 was synergistic in all cell lines with GI50 (micromoles/ml) for Bu decreasing from 67.8 to 45.7 in K562, from 23.3 to 8.0 in HL60, from 46.6 to 36.1 in NB4 and from 34.4 to 17.0 in REH cells. The Combination Index was <1 in all cell lines indicating synergy. Dose Reduction Index, indicating the factor by which the dose of Bu can be decreased to achieve the same treatment effect size, ranged from 1.45 to 3.1. The synergistic effect was greatest in the mismatch repair deficient cell line REH (Combination Index 0.53, Dose Reduction Index 3.1). As expected, the synergistic effect observed did not correlate with increased apoptotic death of leukemic cells implying an alternative mechanism for the derease in proliferation.To our knowledge, this is the first study to show synergy of a clinically available PARP inhibitor with Bu. We believe this data warrants further study with the potential clinical application of increasing the antileukemic effect of stem cell transplantation conditioned with Bu containing preparative regimens. Disclosures:No relevant conflicts of interest to declare.

Abstract A25: PPP2R1A mutations affect PP2A complex formation, and novel protein interactions in gynecological carcinoma cell lines

Abstract Gynecological malignancies (ovarian clear cell and endometrioid carcinomas, endometrial serous, endometrioid and clear cell carcinomas) harbor somatic mutations in the gene PPP2R1A, the scaffolding subunit of the serine/threonine protein phosphatase 2A (PP2A) complex. The PP2A complex is composed of an A (scaffold), C (catalytic) subunit, and different B (regulatory) subunits. The PPP2R1A mutations are proposed to play a role in PP2A B-subunit binding, and thus formation of a functional PP2A phosphatase complex. The aim of this study is to determine how PPP2R1A mutations affect the binding of B-subunits and other novel interactions within the context of ovarian and endometrial carcinomas. The ovarian clear cell line RMG2 and endometrial carcinoma cell lines Hec-1-A and Hec50 all harbor PPP2R1A mutations. The mismatch repair deficient cell line Hec-1-A express a PPP2R1A W257L heterozygous mutation; therefore somatic cell knockout technology was used to generate isogenic PPP2R1A clones that express only mutant PPP2R1A, and clones that express more wild-type than mutant. These Hec-1-A isogenic cell lines were used for PPP2R1A co-immunoprecipiation (Co-IP) followed by MS/MS-SWATHTM analysis to identify PP2A B-subunits and other novel protein interactions. Our preliminary results using Hec-1-A isogenic cell lines suggest that some PP2A B-subunits (PPP2R5D and PPP2R2A) are found to be decreased in abundance in the mutant cell lines. We were able to identify the catalytic subunit (PPP2CA) in PPP2R1A Co-IPs in all cell lines. We also show that PPP2R1A interacts with novel proteins such as histones (Histone 1, Histone H2B type 1-N), ubiquitin ligases (TRIM21) and proteins involved in mitotic control (ANKLE2). Of particular interest we have identified the cancer gene nucleophosmin (NPM1) as a potential novel interactor of PP2A. We have established the isogenic Hec-1-A cell lines as a model system to determine the functional effects of a somatic PPP2R1A mutation. These cell lines have shown that the PPP2R1A mutation affects the ability of some B-subunits to interact and form the PP2A complex. We have also shown that PP2A may interact with novel proteins such as NPM1 and histones. Ongoing work will be completed to determine how these interactions function in the context of ovarian and endometrial carcinomas. Citation Format: Melissa K. McConechy, Vincent Chen, Gregg Morin, James Brenton, David G. Huntsman. PPP2R1A mutations affect PP2A complex formation, and novel protein interactions in gynecological carcinoma cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A25.

Establishment, Characterization and Chemosensitivity of Three Mismatch Repair Deficient Cell Lines from Sporadic and Inherited Colorectal Carcinomas

BackgroundColorectal cancer (CRC) represents a morphologic and molecular heterogenic disease. This heterogeneity substantially impairs drug effectiveness and prognosis. The subtype of mismatch repair deficient (MMR-D) CRCs, accounting for about 15% of all cases, shows particular differential responses up to resistance towards currently approved cytostatic drugs. Pre-clinical in vitro models representing molecular features of MMR-D tumors are thus mandatory for identifying biomarkers that finally help to predict responses towards new cytostatic drugs. Here, we describe the successful establishment and characterization of three patient-derived MMR-D cell lines (HROC24, HROC87, and HROC113) along with their corresponding xenografts.MethodologyMMR-D cell lines (HROC24, HROC87, and HROC113) were established from a total of ten clinicopathological well-defined MMR-D cases (120 CRC cases in total). Cells were comprehensively characterized by phenotype, morphology, growth kinetics, invasiveness, and molecular profile. Additionally, response to clinically relevant chemotherapeutics was examined in vitro and in vivo.Principal FindingsTwo MMR-D lines showing CIMP-H derived from sporadic CRC (HROC24: K-raswt, B-rafmut, HROC87: K-raswt, B-rafmut), whereas the HROC113 cell line (K-rasmut, B-rafwt) was HNPCC-associated. A diploid DNA-status could be verified by flow cytometry and SNP Array analysis. All cell lines were characterized as epithelial (EpCAM+) tumor cells, showing surface tumor marker expression (CEACAM+). MHC-class II was inducible by Interferon-γ stimulation. Growth kinetics as well as invasive potential was quite heterogeneous between individual lines. Besides, MMR-D cell lines exhibited distinct responsiveness towards chemotherapeutics, even when comparing in vitro and in vivo sensitivity.ConclusionsThese newly established and well-characterized, low-passage MMR-D cell lines provide a useful tool for future investigations on the biological characteristics of MMR-D CRCs, both of sporadic and hereditary origin. Additionally, matched patient-derived immune cells allow for comparative genetic studies.

Decreased transcription-coupled nucleotide excision repair capacity is associated with increased p53- and MLH1-independent apoptosis in response to cisplatin

BackgroundOne of the most commonly used classes of anti-cancer drugs presently in clinical practice is the platinum-based drugs, including cisplatin. The efficacy of cisplatin therapy is often limited by the emergence of resistant tumours following treatment. Cisplatin resistance is multi-factorial but can be associated with increased DNA repair capacity, mutations in p53 or loss of DNA mismatch repair capacity.MethodsRNA interference (RNAi) was used to reduce the transcription-coupled nucleotide excision repair (TC-NER) capacity of several prostate and colorectal carcinoma cell lines with specific defects in p53 and/or DNA mismatch repair. The effect of small inhibitory RNAs designed to target the CSB (Cockayne syndrome group B) transcript on TC-NER and the sensitivity of cells to cisplatin-induced apoptosis was determined.ResultsThese prostate and colon cancer cell lines were initially TC-NER proficient and RNAi against CSB significantly reduced their DNA repair capacity. Decreased TC-NER capacity was associated with an increase in the sensitivity of tumour cells to cisplatin-induced apoptosis, even in p53 null and DNA mismatch repair-deficient cell lines.ConclusionThe present work indicates that CSB and TC-NER play a prominent role in determining the sensitivity of tumour cells to cisplatin even in the absence of p53 and DNA mismatch repair. These results further suggest that CSB represents a potential target for cancer therapy that may be important to overcome resistance to cisplatin in the clinic.

Effect of H 2O 2 on cell cycle and survival in DNA mismatch repair-deficient and -proficient cell lines

Patients who develop tumors with Lynch syndrome, which is caused by mutational inactivation of the DNA mismatch repair (MMR) system, have a relatively favorable prognosis compared to patients who develop sporadic tumors. Paradoxically, DNA MMR-deficient cells are resistant to many chemotherapeutic agents, and are capable of bypassing the G2/M checkpoint in vitro. Colon cancers that develop in the setting of Lynch syndrome show an abundant recruitment of immune cells into tumor tissues, which might be expected to increase oxyradical formation, and make the tumor cells more vulnerable to cell death. We examined the chemosensitivity and cell cycle response to oxidative stress in several MMR-deficient (HCT116, SW48, and DLD1) and -proficient (CaCo2, SW480, and HT29) colorectal cancer cell lines. H 2O 2 induced a G2/M cell cycle arrest in both MMR deficient and proficient cell lines, however MMR-deficient cell lines were more sensitive to H 2O 2 toxicity, and the response was more prolonged in MMR-deficient cells. Interestingly, human MutL-homologue ( hMLH1-)defective HCT116 and hMLH1-restored HCT116+ch3 cell lines responded to H 2O 2 with the same degree of G2/M arrest. The survival response of HCT116+ch3 was nearly identical to that of hMLH1-defective HCT116+ch2, although better than the response observed in HCT116 cells. In conclusion, greater cellular sensitivity and G2/M arrest in response to oxidative stress in MMR-deficient colorectal cancer cells could be one of the reasons for the more favorable prognosis seen in patients with Lynch syndrome. However, this sensitivity appears not to be a direct result of a deficient MMR function, but is more likely attributable to spectrum of target gene mutations that occurs in MMR-deficient tumors.

Acyl derivatives of demethylpenclomedine, an antitumor-active, non-neurotoxic metabolites of penclomedine.

The purpose of this investigation was to compare the antitumor activities of a series of acyl derivatives of 4-demethylpenclomedine (DM-PEN), the major plasma metabolite of penclomedine (PEN) observed to be an active antitumor agent in vivo and non-neurotoxic in a rat model with that of DM-PEN. Acyl derivatives were prepared from DM-PEN and evaluated in vivo against human MX-1 breast tumor xenografts implanted subcutaneously (s.c.) or intracerebrally (i.c.). Several derivatives were also evaluated against other human tumor xenografts and murine P388 leukemia cell lines. Several of the acyl derivatives were found to be superior to DM-PEN against MX-1, human ZR-75-1 breast tumor, human U251 CNS tumor and the P388 leukemia parent cell line and lines resistant to cyclophosphamide and carmustine. 4-Demethyl-4-methoxyacetylpenclomedine showed inferior activity to current clinical brain tumor drugs against a glioma cell line, superior activity to temozolomide and procarbazine against the derived mismatch repair-deficient cell line, and superior activity to cyclophosphamide and carmustine but inferior activity to temozolomide against two ependymoma cell lines, all of which were implanted s.c. Proposed mechanisms of activation and action of DM-PEN and the acyl derivatives support the potential clinical superiority of the acyl derivatives.

The cellular mismatch repair system is able to repair mismatches within MLV retroviral double-stranded DNA at a low frequency.

Eukaryotic cells possess several distinct mismatch repair pathways. A mismatch can be introduced in retroviral double-stranded DNA by a pre-existing mutation within the primer binding site (PBS) of the viral RNA genome. In order to evaluate mismatch repair of retroviral double-stranded DNA, Moloney leukemia virus (MLV)-based vectors with a mutation in their PBS were used to infect mismatch repair-competent as well as mismatch repair-deficient cell lines. If the target cells were capable of repairing the mismatch before an infected cell divided, the mismatch within the PBS could be repaired to the wild-type or mutant PBS. If the target cells were unable to repair the mismatch, half the cells in the colony should contain the mutant PBS while the other half should contain the wild-type PBS. To evaluate these predictions, individual colonies were isolated and analyzed by PCR. Almost all mismatch-deficient cell colonies analyzed (cell lines HCT 116 and PMS2-/-) contained both the wild-type and mutated PBS, therefore, mismatches within retroviral double-strand DNA could not be repaired by the mismatch-deficient cells. In contrast, mismatches in approximately 25% of the mismatch repair-competent cell clones analyzed (cell lines HeLa and PMS2+/+) were repaired, while 75% were not. Therefore, the cellular mismatch repair system is able to repair mismatches within viral double-stranded DNA, but at a low frequency.

Identification of hMutLβ, a Heterodimer of hMLH1 and hPMS1

hMLH1 and hPMS2 function in postreplicative mismatch repair in the form of a heterodimer referred to as hMutLalpha. Tumors or cell lines lacking this factor display mutator phenotypes and microsatellite instability, and mutations in the hMLH1 and hPMS2 genes predispose to hereditary non-polyposis colon cancer. A third MutL homologue, hPMS1, has also been reported to be mutated in one cancer-prone kindred, but the protein encoded by this locus has so far remained without function. We now show that hPMS1 is expressed in human cells and that it interacts with hMLH1 with high affinity to form the heterodimer hMutLbeta. Recombinant hMutLalpha and hMutLbeta, expressed in the baculovirus system, were tested for their activity in an in vitro mismatch repair assay. While hMutLalpha could fully complement extracts of mismatch repair-deficient cell lines lacking hMLH1 or hPMS2, hMutLbeta failed to do so with any of the different substrates tested in this assay. The involvement of the latter factor in postreplicative mismatch repair thus remains to be demonstrated.

Sequence-dependent mutations in a shuttle vector plasmid replicated in a mismatch repair deficient human cell line.

We utilized a shuttle vector plasmid (pLSC) to assess the role of DNA sequence and mismatch repair on mutagenesis in human cells. pLSC contains an interrupted 29 bp mononucleotide poly(G) run within a bacterial suppressor tRNA gene, which acts as a highly sensitive mutagenic target for detection of base substitution and frameshift mutations. The frequency of spontaneous mutations in pLSC was found to be similar after replication in either the hMSH6 (GT binding protein) mismatch repair-deficient MT1 line or its parental, mismatch repair-proficient line, TK6. However, the classes of plasmid mutations showed distinct differences in the two cell lines. Single base deletions comprised 48% of the mutations in the 56 independent pLSC plasmids sequenced from MT1 cells while these represented only 18% of the 40 independent pLSC mutants sequenced from the wild-type TK6 cells (P = 0.001). Virtually all the deletions included the mononucleotide run. In contrast, in pSP189, which contains the unmodified supF tRNA without the mononucleotide sequence, no single base deletions were observed for either cell line (P < 0.001). UV treatment of pLSC and pSP189 resulted in a 12-140-fold increase in mutations in TK6 and MT1 cells. These were predominately single base substitution mutations without a large increase in deletion mutations in the mononucleotide run in pLSC. These data indicate that a mononucleotide poly(G) run promotes single base deletion mutations. This effect is enhanced in a hMSH6 mismatch repair-deficient cell line and is independent of UV-induced mutagenesis.

Conditional mutator phenotypes in hMSH2-deficient tumor cell lines.

Two human tumor cell lines that are deficient in the mismatch repair protein hMSH2 show little or no increase in mutation rate relative to that of a mismatch repair-proficient cell line when the cells are maintained in culture conditions allowing rapid growth. However, mutations accumulate at a high rate in these cells when they are maintained at high density. Thus the mutator phenotype of some mismatch repair-deficient cell lines is conditional and strongly depends on growth conditions. These observations have implications for tumor development because they suggest that mutations may accumulate in tumor cells when growth is limited.