[Lipid profile and cardiovascular risk of participants in the lipid measurement program in the industrial park in Höchst]. / Lipidprofil und kardiovaskuläres Risiko der Teilnehmer an den Lipidmesstagen im Industriepark Höchst.

BACKGROUND: Cardiovascular (CV) diseases are still the most frequent cause of death in industrial nations. Employer-initiated screening of the CV risk could make an early contribution to optimization of the prevention strategies. METHODS: In a cross-sectional study the CV risk profile (e.g., dyslipidemia, hypertension, smoking, diabetes mellitus and familial disposition) of 1436 employees at the industrial park in Frankfurt Höchst was analyzed. The total risk was estimated using the PROCAM score. RESULTS: A hypercholesterolemia (low-density lipoprotein, LDL >130 mg/dl) was detected in 36% of the participants. Of the high-risk participants (myocardial infarct, apoplexy and/or diabetes) 23.7% (n = 9/38) were in the target range for LDL as defined by the European Society of Cardiology (ESC) of below 70 mg/dl, 18.4% (n = 7) had levels between 70 and 100 mg/dl and 57.9% (n = 22) had levels of more than 100 mg/dl. In addition, more than half of the subjects (53.2%) had increased blood pressure values (defined as systolic blood pressure ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg). The prevalence of diabetes (blood sugar >126 mg/dl) was very low (1.3%) as was the frequency of manifest CV diseases (1.4% myocardial infarct or apoplexy, 2.9% stabile angina pectoris or peripheral arterial occlusive disease, PAOD). CONCLUSION: The data confirm that the risk factors high blood pressure and dyslipidemia are widespread and the achievement of target values is insufficient, particularly with a high risk of CV. Behavioral therapeutic and/or pharmaceutical measures should be instigated in order to better exploit the high preventive potential for carriers of these risk factors.

Transcatheter Atrial Septal Defect Closure in Children with and without Fluoroscopy: A Comparison.

OBJECTIVE: The aim of this study was to compare feasibility, effectiveness, safety, and outcome of atrial septal defect (ASD) device closure in children with and without fluoroscopy guidance. METHODS AND RESULTS: Children undergoing transcatheter ASD closure between 2002 and 2016 were included into this single center, retrospective study. Patients were analysed in two groups [1: intraprocedural fluoroscopy ± transoesophageal echocardiography (TOE) guidance; 2: TOE guidance alone]. Three-hundred-ninety-seven children were included, 238 (97 male) in group 1 and 159 (56 male) in group 2. Two-hundred-twenty-nine of 238 (96%) patients underwent successful fluoroscopy guided ASD closures versus 154/159 (97%) successful procedures with TOE guidance alone. Median weight (IQR) at intervention was 20kg (16.0-35.0) in group 1 versus 19.3kg (16.0-31.2) in group 2. Mean (SD) preinterventional ASD diameter was 12.4mm (4.4) in group 1 versus 12.2mm (3.9) in group 2. There was no significant difference in number of defects or characteristics of ASD rims. Median procedure time was shorter in group 2 [60min (47-86) versus 34min (28-44)]. Device-size-to-defect-ratio was similar in both groups [group 1: 1.07 versus group 2: 1.09]. There were less technical intraprocedural events in group 2 [10 (6.3%) versus 47 (20%)]. Intraprocedural complications were less frequent in group 2 [1 (0.6%) versus 8 (3.3%)]. CONCLUSION: Transcatheter ASD device closure with TOE guidance alone (i.e., without fluoroscopy) is as effective and safe as ASD closure with fluoroscopy guidance. As fluoroscopy remains an important adjunct to transoesophageal echocardiography, especially in complex defects and complications, procedures are always performed in a fully equipped cardiac catheterization laboratory.

Use of coffee, caffeinated drinks and caffeine tablets for cognitive enhancement in pupils and students in Germany.

INTRODUCTION: Substance use for cognitive enhancement (CE) is a topic of increasing importance. There are only few data about substances, prevalence rates and factors associated with CE. The aim of this study was to assess first data about the use of coffee, caffeinated drinks and caffeine tablets for CE at school and university. METHODS: A self-report questionnaire was developed to analyze 1 547 pupils and students about their use of coffee, caffeine tablets, and caffeinated drinks for CE and factors associated with this use. RESULTS: Lifetime, past-year, and past-month prevalence for the use of coffee for CE was 53.2%, 8.5%, and 6.3%, for the use of caffeinated drinks 39%, 10.7%, and 6.3%, and for the use of caffeine tablets 10.5%, 3.8%, and 0.8%. Use of caffeinated substances for CE was influenced by gender and school grades. DISCUSSION: The use of coffee and caffeinated drinks for CE was found to be widespread in the surveyed population. Although the use of caffeine tablets was found to be smaller than the above-mentioned means, it still indicates a relatively high disposition for using tablets for purposes of CE.

[Lung function testing in children before and after an age-adapted SCUBA dive in a swimming pool]. / Lungenfunktionsmessung bei Kindern vor und nach einem altersadaptierten SCUBA Tauchgang im Schwimmbad.

BACKGROUND: The number of children that SCUBA dive is increasing. Airway narrowing while SCUBA diving can cause dangerous complications like pulmonary barotrauma and arterial gas embolism. Statistics show that children are at an increased risk. Since data are scarce, the goal of this study was to gain new knowledge about acute lung function changes in children while SCUBA diving. MATERIAL AND METHODS: 41 children aged 8 - 14 years underwent lung function testing (spirometry and residual volume measurement) before and after a single age-adapted SCUBA dive in a swimming pool. RESULTS: A significant reduction of the dynamic expiratory lung function parameters FEV (1) (p < 0.01), FEV (1)/VC (p < 0.05), MEF 75 % (p < 0.05), MEF 50 % (p < 0.01) und MEF 25 % (p < 0.05) was measured. No statistically significant change of the residual volume was found. A decrease of FEV (1) > 10 % (12 % - 21 %) was found in 5 children (12.2 %). CONCLUSION: The majority of the children (87.8 %) did not show any relevant lung function changes. Five children had a considerable reduction of FEV (1). Signs indicate the importance of bronchial hyperreactivity (BHR) as a key factor. Children with asthma or BHR should not SCUBA dive. A detailed medical examination is recommended (including an unspecific bronchial provocation test) before starting to dive.

Non-medical use of prescription stimulants and illicit use of stimulants for cognitive enhancement in pupils and students in Germany.

INTRODUCTION: The aim of this study was to assess for the first time the prevalence and factors associated with stimulant use exclusively for cognitive enhancement among pupils and university students in Germany. METHODS: A sample of 1 035 pupils (vocational and grammar schools) in small and big cities and 512 university students of 3 Departments (Medicine, Pharmacy, Economics) completed a questionnaire regarding knowledge and use of stimulants for cognitive enhancement and factors associated with their use. RESULTS: Lifetime prevalence for use of prescription stimulants (methylphenidate, amphetamines) for cognitive enhancement in pupils was 1.55% and in students 0.78%. Last-year and last-month prevalence rates were significantly lower. 2.42% of pupils and 2.93% of students reported lifetime illicit use of stimulants (amphetamines, cocaine, ecstasy) for cognitive enhancement with lower last-year and last-month rates. Prevalence was higher in male pupils, pupils from vocational schools and pupils with bad marks. DISCUSSION: The illicit use of stimulants for cognitive enhancement is significantly higher than non-medical use of prescription stimulants among pupils and students. Stimulant use is determined by gender, school type, and school marks. The potential risks associated with stimulant use require early awareness and intervention strategies.

[The repeatability of forced expiratory manoeuvres in 4- to 6-year-old children with intermittent bronchial asthma in healthy and in exacerbated status]. / Reproduzierbarkeit der FEV1 bei Kindern von 4 bis 6 Jahren mit intermittierendem Asthma bronchiale im beschwerdefreien sowie im Beschwerdeintervall.

INTRODUCTION: The question about the repeatability of forced expiratory manoeuvres in childhood lung function testing is of scientific and clinical interest. The following study investigated to what extent children ≥ 4 to < 7 years of age with intermittent bronchial asthma are able to produce reproducible lung function measurements on the one hand in the healthy status and on the other hand in an exacerbated status. METHOD: 64 children at the age of ≥ 4 to < 7 years with intermittent preschool bronchial asthma performed lung function measurements in the healthy status and again in an exacerbated status. FEV (1) values from the measurements were analysed according to ATS/ERS guidelines concerning repeatability. RESULTS: According to the new ATS/ERS guidelines 74.6 % of the children could perform at least 2, and 59.3 % could perform 3 repeatable measurements in the healthy status. In the exacerbated status this was 87.5 % and 68.8 %, respectively. There were no significant differences between the healthy and the exacerbated status and between the age groups. Compared to former repeatability criteria, children of this age group can perform significantly more reproducible measurements (p < 0.0001). CONCLUSION: The ATS/ERS guidelines from 2007 simplify the repeatability of forced expiratory manoeuvres in children at ≥ 4 to < 7 years of age compared to the former criteria. Repeatability is not reduced in the exacerbated status. 74.6 % of children in this age group can produce repeatable lung function measurements.

Temozolomide- and fotemustine-induced apoptosis in human malignant melanoma cells: response related to MGMT, MMR, DSBs, and p53.

Malignant melanomas are highly resistant to chemotherapy. First-line chemotherapeutics used in melanoma therapy are the methylating agents dacarbazine (DTIC) and temozolomide (TMZ) and the chloroethylating agents BCNU and fotemustine. Here, we determined the mode of cell death in 11 melanoma cell lines upon exposure to TMZ and fotemustine. We show for the first time that TMZ induces apoptosis in melanoma cells, using therapeutic doses. For both TMZ and fotemustine apoptosis is the dominant mode of cell death. The contribution of necrosis to total cell death varied between 10 and 40%. The O(6)-methylguanine-DNA methyltransferase (MGMT) activity in the cell lines was between 0 and 1100 fmol mg(-1) protein, and there was a correlation between MGMT activity and the level of resistance to TMZ and fotemustine. MGMT inactivation by O(6)-benzylguanine sensitized all melanoma cell lines expressing MGMT to TMZ and fotemustine-induced apoptosis, and MGMT transfection attenuated the apoptotic response. This supports that O(6)-alkylguanines are critical lesions involved in the initiation of programmed melanoma cell death. One of the cell lines (MZ7), derived from a patient subjected to DTIC therapy, exhibited a high level of resistance to TMZ without expressing MGMT. This was related to an impaired expression of MSH2 and MSH6. The cells were not cross-resistant to fotemustine. Although these data indicate that methylating drug resistance of melanoma cells can be acquired by down-regulation of mismatch repair, a correlation between MSH2 and MSH6 expression in the different lines and TMZ sensitivity was not found. Apoptosis in melanoma cells induced by TMZ and fotemustine was accompanied by double-strand break (DSB) formation (as determined by H2AX phosphorylation) and caspase-3 and -7 activation as well as PARP cleavage. For TMZ, DSBs correlated significantly with the apoptotic response, whereas for fotemustine a correlation was not found. Melanoma lines expressing p53 wild-type were more resistant to TMZ and fotemustine than p53 mutant melanoma lines, which is in marked contrast to previous data reported for glioma cells treated with TMZ. Overall, the findings are in line with the model that in melanoma cells TMZ-induced O(6)-methylguanine triggers the apoptotic (and necrotic) pathway through DSBs, whereas for chloroethylating agents apoptosis is triggered in a more complex manner.

Rechallenge with temozolomide with different scheduling is effective in recurrent malignant gliomas.

Treatment of recurrent malignant glioma, which has a poor patient prognosis, has not been standardised. Moreover, it is unclear whether repeated treatment with temozolomide is effective in patients who received previous temozolomide treatment before developing a recurrence. Here, we present the results of a high-dose individually adapted 21-day regimen demonstrating that rechallenge is effective even in patients expressing O6-methylguanine-DNA methyltransferase (MGMT) in the tumor. Twenty-one patients with recurrent malignant gliomas pre-treated with temozolomide, 18 WHO IV glioblastoma (GBM) and 3 WHO III patients, received 100 mg/m2 temozolomide on days 1-21/28. The GBM patients had a median Karnofsky performance status of 60% and a median age of 54.8 years. Blood counts decreased continuously, enabling a gradual dose adaptation. When blood counts dropped below normal values, temozolomide was applied on days 1-5/7. Dosage was reduced to 50-75 mg/m2 in 11 patients and gradually increased up to 130 mg/m2 in 3 patients. WHO grade 3/4 toxicity was hematological in 3 patients and non-hematological in 3 patients. In GBM patients (n=18), response after >3 months was complete in 3 patients, partial in 1 (22%), stable disease in 7 (39%) and progressive disease in 7 (39%). Progression-free survival at 6 months (PFS-6M) was 39%. Median survival was 9.1 months from relapse and 17.9 months overall. Of the patients with unmethylated MGMT promoter, 2/7 were progression-free for >6 (15 and 19) months. The data indicate that rechallenge with near-continuous, higher-dose temozolomide (100 mg/m2 on days 1-21/28 or days 1-5/7 with individual dose adaptation) is also feasible in patients with critical blood counts. Objective responses can be achieved even after relapse during a conventional 5/28-day regimen. The resistance of tumors characterized by unmethylated MGMT promoter may be overcome by near continuous temozolomide administration, which is probably most effective with a 5/7-day schedule. In spite of the relatively poor clinical prognosis, the data indicate that rechallenge with temozolomide with a dose-dense and long-lasting administration protocol is tolerable and comparable with other reported treatment protocols involving temozolomide.

Mouse embryonic stem cells are hypersensitive to apoptosis triggered by the DNA damage O(6)-methylguanine due to high E2F1 regulated mismatch repair.

Exposure of stem cells to genotoxins may lead to embryonic lethality or teratogenic effects. This can be prevented by efficient DNA repair or by eliminating genetically damaged cells. Using undifferentiated mouse embryonic stem (ES) cells as a pluripotent model system, we compared ES cells with differentiated cells, with regard to apoptosis induction by alkylating agents forming the highly mutagenic and killing DNA adduct O(6)-methylguanine. Upon treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ES cells undergo apoptosis at much higher frequency than differentiated cells, although they express a high level of the repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Apoptosis induced by MNNG is due to O(6)-methylguanine DNA adducts, since inhibition of MGMT sensitized ES cells. The high sensitivity of ES cells to O(6)-methylating agents is due to high expression of the mismatch repair proteins MSH2 and MSH6 (MutSalpha), which declines during differentiation. High MutSalpha expression in ES cells was related to a high hyperphosphorylated retinoblastoma (ppRb) level and E2F1 activity that upregulates MSH2, causing, in turn, stabilization of MSH6. Non-repaired O(6)-methylguanine adducts were shown to cause DNA double-stranded breaks, stabilization of p53 and upregulation of Fas/CD95/Apo-1 at significantly higher level in ES cells than in fibroblasts. The high apoptotic response of ES cells to O(6)-methylguanine adducts may contribute to reduction of the mutational load in the progenitor population.

Organocatalytic syntheses of bioactive natural products.

The current scope and limitations of organocatalytic reactions and the consequences for the strategic planning of natural product syntheses are discussed. Examples from our group include the total synthesis of UCS1025A and lepidopteran sex pheromones.

Apoptosis in UV-C light irradiated p53 wild-type, apaf-1 and p53 knockout mouse embryonic fibroblasts: interplay of receptor and mitochondrial pathway.

Mouse embryonic fibroblasts (MEFs) deficient for the transcription factor p53 are hypersensitive to UV-C light. They also show a reduced recovery from UV-C induced replication blockage and are unable to repair UV-C photoproducts. In this study, we utilized wild-type (wt), Apaf-1 deficient (apaf-1(-/-)) and p53 deficient (p53(-/-)) MEFs in order to elucidate the role of non-repaired UV-C lesions in apoptotic signalling. Corresponding with the cellular sensitivity determined by the WST assay, p53(-/-) cells displayed the highest level of apoptosis, whereas wt cells showed moderate apoptosis after UV-C irradiation. Apaf1(-/-) cells were most resistant. In wt cells apoptosis was executed both via the mitochondrial and the receptor-mediated pathway, as shown by Bcl-2 decline, induction of fasR and activation of caspases-3,8,9. In apaf-1(-/-) (p53(+/+)) cells, the mitochondrial pathway was blocked downstream of Bcl-2, indicating that in this case apoptosis was mediated via the induction of fasR and caspase-3,8 activation. In p53 deficient cells, non-repaired UV-C induced DNA lesions triggered sustained up-regulation of fas ligand (fasL) mRNA, which was not seen in wt and apaf-1(-/-) cells. Therefore, in p53(-/-) MEFs, the receptor/ligand triggered pathway appeared to be dominant. This was confirmed by significant reduction of apoptosis after DN-FADD transfection. As opposed to wt and apaf-1(-/-) cells, p53 deficient MEFs showed no induction of Fas receptor and no Bcl-2 decline. Nevertheless, the resulting caspase-8 and -3 activation was stronger compared to wt and apaf-1(-/-) cells. The data indicate that UV-C light activates in MEFs both the Fas (CD95, Apo-1) receptor and the mitochondrial damage pathways. In p53(-/-) cells, however, the high level of non-repaired DNA damage forces signalling by fasL upregulation, leading to enhanced UV-C-induced apoptosis.

DNA repair in resistance to alkylating anticancer drugs.

The major critical target of alkylating antineoplastic drugs belonging to the group of methylating and chloroethylating agents is DNA. DNA alkylation lesions can be repaired by the action of alkyltransferase (MGMT) and base excision repair enzymes. The major cell killing and apoptotic alkylation lesions are O6-methylguanine (O6MeG) and O6-chloroethylguanine. O6MeG causes mispairing with thymine which is erroneously processed by mismatch repair (MMR), leading to secondary lesions that potently trigger the mitochondrial apoptotic pathway. Apoptosis induced by O6MeG is a late cellular response that requires cell proliferation to occur. Data are available indicating that DNA double-strand breaks are actively involved as the ultimate trigger of apoptosis. O6MeG and O6-chloroethylguanine are repaired by the specific action of MGMT thus counteracting the killing effects of the lesions. The expression of MGMT is highly variable and is often increased in tumors compared to normal tissue. Determination of MGMT activity in various tumors showed low expression in brain, pancreas and skin and high expression in testicle, breast, colorectal, lung and ovarian tumors. Distribution profiles of MGMT revealed non-random distribution indicating the existence of subpopulations exhibiting low and high activity. Since MGMT is one of the most important factors determining drug resistance to alkylation, strategies have been developed to inhibit MGMT in tumors with the aid of MGMT inhibitors and overexpression of MGMT in healthy, non-target tissue (e.g. blood stem cells) by transferring a mutated form of MGMT inaccessible to inhibition. Targeting MGMT inhibitors to tumors may further enhance the antineoplastic efficiency of alkylating agents. The role of base excision repair, Fos and p53 in drug resistance to alkylation is also discussed.

[Infection with fasciola hepatica causing elevated liver-enzyme results and eosinophilia - serologic and endoscopic diagnosis and therapy]. / Fasciola hepatica als Ursache einer Leberwerterhöhung mit Eosinophilie - serologische und endoskopische Diagnostik und Therapie.

A 36 year old man was diagnosed with eosinophilia and elevated liver-enzyme levels in a preoperative routine laboratory testing. In the course of the evaluation we found high levels of Fasciola hepatica specific antibodies which lead to an ERC with biliary aspiration after choleretic stimulation. The bile showed Fasciola eggs and subsequently we recovered a parasite from the bile duct. Abdominal CT- and MRI-Scans showed lesions of the liver consistent with hepatic fascioliasis. Focussed patient's history revealed ingestion of contaminated watercress in Turkey as the most likely source of infection. For patients with elevated liver-enzymes of unknown etiology with eosinophilia or in combination with a congruent patient's history even without eosinophilia serologic studies of Fasciola hepatica seem to be advisable. We suggest an ERC with biliary aspiration to prove the diagnosis in case of positive results, if the prepatency period of 4 months after the ingestion is over. Fasciola hepatica is rarely diagnosed in Germany probably due to the lack of awareness of the disease.

Rapid access to polyketide scaffolds via vinylogous Mukaiyama aldol reactions.

[reaction: see text]. The use of the Z-configured vinylogous silyl ketene acetals in Mukaiyama aldol reactions is described. Isopropyl alcohol as scavanger and the use of tris(pentafluorophenyl)borane as the Lewis acid are required for obtaining the gamma-alkylated syn-product selectively. In cases of alpha-chiral aldehydes, Felkin-Anh selectivity was observed.

BER, MGMT, and MMR in defense against alkylation-induced genotoxicity and apoptosis.

Methylating carcinogens and cytostatic drugs induce different methylation products in DNA. In cells not expressing the repair protein MGMT or expressing it at a low level, O6-methylguanine is the major genotoxic, recombinogenic, and apoptotic lesion. Genotoxicity and apoptosis triggered by O6-methylguanine require mismatch repair (MMR). In cells expressing O6-methylguanine-DNA methyl transferase (MGMT) at a high level or for agents producing low amounts of O6-methylguanine, N-alkylations become the major genotoxic lesions. N-Alkylations are repaired by base excision repair (BER). In mammalian cells, naturally occurring mutants of BER have not been detected, which points to the importance of BER for viability. In order to ascertain the role of BER in cellular defense, BER was modulated either by transfection or mutational inactivation. It has been shown that overexpression of N-methylpurine-DNA glycosylase (MPG) does not protect, but rather sensitizes cells to SN2 agents. This has been interpreted in terms of an imbalance in BER. Regarding abasic site endonuclease (APE), transient but not stable overexpression of the enzyme was achieved upon transfection in CHO cells, which indicates that unphysiologic APE levels are not tolerated by the cell. Besides the repair function, APE (alias Ref-1) exerts redox capability by which the activity of various transcription factors is modulated. Therefore, it is possible that stable overexpression of mammalian APE impairs transcriptional regulation of genes, whereas transient overexpression may exert some protective effect. DNA polymerase beta (Pol beta) transfection was ineffective in conferring resistance to methylmethane sulfonate (MMS). On the other hand, Pol beta-deficient cells proved to be highly sensitive to methylation-induced chromosomal aberrations and reproductive cell death. The dramatic hypersensitivity in the killing response is largely due to induction of apoptosis. Obviously, nonrepaired BER intermediates are clastogenic and act as a strong trigger of the apoptotic pathway. The elements of this pathway are currently under investigation.

Acquired resistance of melanoma cells to the antineoplastic agent fotemustine is caused by reactivation of the DNA repair gene MGMT.

Acquired resistance to antineoplastic agents is a frequent obstacle in tumor therapy. Malignant melanoma cells are particularly well known for their unresponsiveness to chemotherapy; only about 30% of tumors exhibit a transient clinical response to treatment. In our study, we investigated the molecular mechanism of acquired resistance of melanoma cells (MeWo) to the chloroethylating drug fotemustine. Determination of O(6)-methylguanine-DNA methyltransferase (MGMT) activity showed that MeWo cells that acquired resistance to fotemustine upon repeated treatment with the drug display high MGMT activity, whereas the parental cell line had no detectable MGMT. The resistant cell lines exhibit cross-resistance to other O(6)-alkylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine. Acquired resistance to fotemustine was alleviated by treatment with the MGMT inhibitor O(6)-benzylguanine demonstrating that reactivation of MGMT is the main underlying cause of acquired alkylating drug resistance. As compared with control cells, both MGMT mRNA and MGMT protein were expressed at a high level in fotemustine resistant cells. Southern blot analysis proved that the MGMT gene was not amplified. There was also only an insignificant difference in the CpG methylation pattern of the MGMT promoter whereas a clear hypermethylation in the body of the gene was observed in fotemustine resistant cells. The conclusion that hypermethylation is responsible for reactivation of the MGMT gene gained support by the finding that MGMT activity significantly declined and cells reverted (partially) to the parental sensitive phenotype upon treatment with 5-azacytidine. This is the first report of acquired resistance to a chloroethylating antineoplastic drug of melanoma cells due to gene hypermethylation.

The first total synthesis of +-ratjadone.

The first total synthesis of ratjadone was achieved using a highly convergent approach joining three subunits together with a Wittig olefination and a selective Heck reaction as the pivotal steps. Besides establishing a robust and reliable route for the synthesis of this orphan ligand, the configuration of unknown stereocenters could also be determined. This synthesis not only provides an additional access to a biologically important compound but also enables the synthesis of structural analogues for biological target identification.

Cloning and functional analysis of cDNA encoding the hamster Bcl-2 protein.

We have cloned cDNA encoding hamster Bcl-2 protein from total RNA of CHO-9 cells by RT-PCR using oligonucleotide primers sharing homology with the sequence of mouse and rat bcl-2. The fragments spanning the total coding region were cloned into pCR4-TOPO and sequenced for verification. The hamster bcl-2 cDNA has a size of 711 nucleotides and encodes a polypeptide of 236 amino acids. Hamster Bcl-2 shares 95.8 and 88.6% similarity with mouse and human Bcl-2, respectively. Northern blot analysis revealed a single 7.5 kb bcl-2 transcript in hamster (CHO-9), mouse (BK4), and rat (H5) cells and a 8.5 kb bcl-2 mRNA in human (HeLa MR) cells. The bcl-2 cDNA (771 bp) was recloned into pcDNA3 and the recombinant construct was transiently transfected into MGMT-deficient HeLa MR cells. Expression of hamster Bcl-2 rendered the cells more resistant to MNNG-induced cytotoxicity, which is consistent with the anti-apoptotic function of Bcl-2.

Nuclear translocation of mismatch repair proteins MSH2 and MSH6 as a response of cells to alkylating agents.

Mammalian mismatch repair has been implicated in mismatch correction, the prevention of mutagenesis and cancer, and the induction of genotoxicity and apoptosis. Here, we show that treatment of cells specifically with agents inducing O(6)-methylguanine in DNA, such as N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea, elevates the level of MSH2 and MSH6 and increases GT mismatch binding activity in the nucleus. This inducible response occurs immediately after alkylation, is long-lasting and dose-dependent, and results from translocation of the preformed MutSalpha complex (composed of MSH2 and MSH6) from the cytoplasm into the nucleus. It is not caused by an increase in MSH2 gene activity. Cells expressing the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), thus having the ability to repair O(6)-methylguanine, showed no translocation of MutSalpha, whereas inhibition of MGMT by O(6)-benzylguanine provoked the translocation. The results demonstrate that O(6)-methylguanine lesions are involved in triggering nuclear accumulation of MSH2 and MSH6. The finding that treatment of cells with O(6)-methylguanine-generating mutagens results in an increase of MutSalpha and GT binding activity in the nucleus indicates a novel type of genotoxic stress response.