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Biomedical subjects

R Lewensohn

Publications and source records attributed to R Lewensohn.

At least 37 records · Page 2Linked to original sources
PubMed1999

Chromosomal sensitivity to X-ray irradiation during the G2 phase in lymphocytes of patients with hereditary cutaneous malignant melanoma as compared to healthy controls.

Recent reports have suggested that elevated chromosomal aberration yields following X-ray irradiation of skin fibroblasts and peripheral lymphocytes in the G2 phase of the cell cycle are characteristic of affected members of cancer-prone families. These studies propose that the phenomenon is a consequence of impaired caffeine- and arabinofuranosylcytosine (ara-C)-sensitive DNA repair and might be a useful indicator of genetic susceptibility to cancer. We have tested G2 chromosomal X-ray sensitivity in peripheral blood lymphocytes from members of kindreds with hereditary cutaneous malignant melanoma (HCMM) combined with the dysplastic nevus syndrome (DNS), disorders in which susceptibility to skin cancer is inherited in an autosomal dominant pattern. In the assay lymphocytes from patients with HCMM/DNS exhibited responses indistinguishable from normal healthy controls. Furthermore, the radiation-induced aberration yields were potentiated to the same strong extent by post-treatments with caffeine, or a combination of ara-C and hydroxyurea, both in lymphocytes from individuals with HCMM/DNS and lymphocytes from healthy controls. Thus, lymphocytes of affected patients with HCMM/DNS do not have an increased sensitivity to X-ray irradiation in the G2 phase of the cell cycle.

Adult↗
PubMed1999

DNA double-strand break repair, DNA-PK, and DNA ligases in two human squamous carcinoma cell lines with different radiosensitivity.

PURPOSE: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs). DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation. METHODS AND MATERIALS: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE). DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham). DNA ligase activity in crude cell extracts was measured using [5'-33P] Poly (dA) x (oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting. RESULTS: Applying the commonly used linear-quadratic equation to describe cell survival, S = e-alphaD-betaD2, the two cell lines roughly have the same alpha value (approximately 0.40 Gy(-1)) whereas the beta value was considerably higher in UM-SCC-14A (0.067 Gy(-2)+/-0.007 Gy(-2) [SEM]) as compared to UM-SCC-1 (0.013 Gy(-2)+/-0.004 Gy(-2) [SEM]). Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE. The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A ( < 0.05). The constitutive level of DNA ligase activity was similar in the two cell lines. CONCLUSIONS: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity.

Carcinoma, Squamous Cell↗
PubMed1999

DNA-dependent protein kinase content and activity in lung carcinoma cell lines: correlation with intrinsic radiosensitivity.

Intrinsic radiosensitivity and rejoining of radiation-induced DNA double-strand breaks (DNA-dsb) were analysed in five lung carcinoma cell lines: U-1285, U-1906, H-69, H-82 and U-1810. RS correlated with both the initial phase of DNA-dsb rejoining, at 15 min (r2 = 0.818) and the late phase, at 120 min postirradiation (r2 = 0.774), the most sensitive cell line (U-1285) showing least dsb rejoining and the most resistant (U-1810) showing most dsb rejoining of all five cell lines studied. As DNA-PK has been recognised as an important molecular component involved in DNA-dsb repair, we analysed content and activity of this kinase. We found that DNA-PK content and activity correlated with RS (r2 = 0.941 and r2 = 0.944, respectively). The lowest DNA-dependent content/activity was found in the most radiosensitive cells, U-1285 and H-69, whilst the highest content/activity was found in the most radioresistant cells U-1810. These results suggest a correlation between RS and DNA-PK content/activity in lung carcinoma cell lines.

Blotting, Western↗
PubMed1999

Cell cycle-dependent regulation of the DNA-dependent protein kinase.

Human DNA-dependent protein kinase (DNA-PK) is a nuclear-localized serine/threonine protein kinase. The holoenzyme consists of a catalytic subunit with a molecular mass of 465 kDa and a DNA-binding heterodimer Ku86/70. The kinase has been implicated in a variety of nuclear processes including V(D)J recombination, double-strand break repair, and transcription. Cells with defective DNA-PK activity show increased radiosensitivity and lack of V(D)J recombination. To study DNA-PK activity during the cell cycle, HeLa cells were separated by elutriation centrifugation into different cell cycle compartments based on cellular size. DNA-PK activity was found to vary during the cell cycle. The kinase activity was lowest during G1 phase and increased dramatically as the cells entered S phase and remained high during the G2-phase. The subcellular distribution of DNA-PKcs is relocalized from the cytoplasm during M and G1 phases to the nucleus during G1-S phase transition and S phase. Expression of both the catalytic subunit and the Ku86/70 heterodimer was found to be constant throughout the cell cycle. This study demonstrates that DNA-PK activity as well as its subcellular localization fluctuates during the cell cycle. In addition, the distribution of DNA-PK during M phase corresponds with low DNA-PK activity.

Blotting, Western↗
PubMed1999

Epstein-Barr virus-transformed lymphoblastoid cell lines of ataxia telangiectasia patients are defective in X-ray-induced apoptosis.

PURPOSE: To investigate and compare the propensity of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) obtained from unaffected healthy individuals and ataxia telangectasia (A-T) patients to undergo apoptosis after X-ray exposure. MATERIAL AND METHODS: The LCL were exposed to 1-4 Gy X-rays at a dose-rate of 1.36 Gy/min. At various post-irradiation times (0, 24, 48 and 72 h) the induction of apoptosis was analysed by: (1) monitoring the formation of high molecular weight (HMW) DNA fragments by field inversion pulse gel electrophoresis (FIGE); and (2) morphological characterization of apoptotic cells after fluorescence staining. In parallel, cell-cycle distribution, monitored by DNA flow cytometry, was investigated in these cells. RESULTS: The LCL obtained from the A-T homozygotes were resistant to undergoing radiation-induced apoptosis during the observation time used. On the contrary, LCL from unaffected healthy controls displayed significant radiation-induced chromatin fragmentation seen at 48 h and 72 h after irradiation. In these cells, radiation-induced G -arrest (24h post-irradiation) preceded chromatin cleavage. In A-T LCL, the defective G1-arrest was not followed by apoptosis. CONCLUSIONS: In spite of a defective cell-cycle control, EBV-transformed LCL of A-T patients compared with unaffected healthy controls do not undergo X-ray-induced apoptosis, at least during their first post-irradiation cell cycle.

Apoptosis↗
PubMed1999

In vitro cellular accumulation and cytotoxicity of liposomal and conventional formulations of daunorubicin and doxorubicin in resistant K562 cells.

Previous investigations have indicated the possibility to circumvent multidrug resistance (MDR) by incorporation of an anthracycline into liposomes. We examined the in vitro cytotoxicity and cellular drug accumulation of the anthracyclines daunorubicin and doxorubicin compared with the commercially available liposomal formulations DaunoXome and Caelyx in human myelogenous leukemia K562 cells. The drug-sensitive parental K562/K line was compared with the P-glykoprotein (P-gp)-expressing cell lines K562/Dnr and K562/Vcr. Two cell lines with reduced levels of topoisomerase II (K562/Nov and K562/Ida) were also included. The cytotoxicity was determined by fluorometric microculture cytotoxicity assay and the cellular drug levels were determined by high performance liquid chromatograghy. There was a strong inverse correlation between P-gp levels and cellular drug accumulation (rho = -0.83, p = 0.04) and cytotoxicity (rho = -0.95, p = 0.01) of daunorubicin. Also the cytotoxicity of DaunoXome and doxorubicin was related to P-gp levels (rho = -0.96, p = 0.01 and rho = -0.90, p = 0.07, respectively). Caelyx did not show any cytotoxic effect due to impaired cellular uptake of the pegylated liposome. Regardless of the P-gp levels of the treated cells, DaunoXome showed the same cytotoxic effect despite lower intracellular accumulation (range 22-47%), compared with conventional daunorubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

The kinetics and cytotoxicity of cisplatin and its monohydrated complex.

This paper examines the monohydrated complex of cisplatin (MHC) with respect to kinetics and cytotoxicity. Equilibrium mixtures of cisplatin and hydrated species have been used in previous studies of a similar nature. To our knowledge, this is the first paper examining MHC after isolation and quantification. This was accomplished using liquid chromatography with porous graphitic carbon. MHC and cisplatin were quantified over time in a suspension of the small-cell lung cancer cell line U-1285. Cytotoxicity was evaluated using the fluorescent microculture cytotoxicity assay. MHC was significantly more cytotoxic than cisplatin at the high end of the drug concentrations tested. In culture media with low chloride ion concentrations, the stability of MHC was related to changes in pH. At a pH of between 6.0 and 7.2, MHC was rapidly converted to cisplatin. In culture media with a pH above 7.2, MHC was considerably more stable. These findings might have clinical significance given that MHC circulates in the blood stream of patients receiving cisplatin infusions and that solid tumours often have environments that are extremely acidotic.

Antineoplastic Agents↗
PubMed1998

Higher spontaneous apoptotic index in small cell compared with non-small cell lung carcinoma cell lines; lack of correlation with Bcl-2/Bax.

Spontaneous apoptosis was assessed in ten small-cell (SCLC) and five non-small cell (NSCLC) lung carcinoma cell lines by the TUNEL assay and chromatin cleavage. TUNEL staining showed significantly higher apoptotic index (AI) in SCLC (2-20%) compared with NSCLC lines (0.2-1%) in untreated exponentially growing cells. Six out of ten SCLC and none of the NSCLC showed DNA fragmentation when analysed by agarose gel electrophoresis. Field inversion pulse gel electrophoresis was used in a subset of cell lines and showed the presence of high molecular weight fragments in untreated SCLC lines U-1285 and U-1906 cells, but not in the NSCLC line U-1810. Important molecular determinants of apoptosis were studied by Western blot. Bcl-2 was detected at highest level in SCLC. There was no correlation between the ratio Bcl-2/Bax and AI in all tested cell lines. Neither p53 nor c-Myc protein status correlated to AI. Pro-caspase-3 was expressed in all cell lines without correlation to AI and no difference between the SCLC and NSCLC groups was found. In conclusion, this study shows a high degree of spontaneous apoptosis in SCLC lines compared to NSCLC lines unrelated to Bcl-2/Bax ratio.

Apoptosis↗
PubMed1998

Comparison between radiation-induced cell cycle delay in lymphocytes and radiotherapy response in head and neck cancer.

A study was made evaluating the use of radiation-induced cell cycle delay in lymphocytes to predict tumour response to radiotherapy. Peripheral blood lymphocytes were isolated from whole blood from 49 patients with head and neck cancer before treatment with radiotherapy and from 25 healthy donors. The clinical response to radiotherapy was assessed at 0-2 months after treatment. The level of radiation-induced cell cycle delay was measured using flow cytometry after mitogen stimulation of lymphocytes. The analysis of ten normal donors gave no significant difference in variability between the intra-assay and the intra-donor samples. However, the cell cycle data for lymphocytes from these healthy donors showed significant inter-individual differences in G2 phase accumulation. Patients showing no response to radiotherapy had a high level of S-phase cells compared with partial (P < 0.001) and complete responders (P = 0.016). An inverse relationship was found when analysing the fraction of cells in G2 (P = 0.009 and 0.034 respectively). In general, healthy donors had similar cell cycle kinetics compared with the non-responders. In conclusion, the result indicates that radiation-induced cell cycle delay in lymphocytes is inversely correlated with tumour response to radiotherapy in head and neck cancer patients. However, the value of the present test for predicting individual tumour response is limited, because of assay variability and overlap between groups.

Adult↗
PubMed1998

Comparison of the cytotoxic activity of melphalan with L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine in human tumour cell lines and primary cultures of tumour cells from patients.

m-L-sarcolysin (m-L-SL) is an isomer of melphalan (Mel) with the di(2-chloroethyl) amino group being substituted in the meta position of phenylalanine. By covalent conjugation of amino acids to m-L-SL, a peptide complex consisting of six m-L-SL-based oligopeptides known as peptichemio (PTC) was developed previously. In the present study, the cytotoxic activity pattern of the different oligopeptides of PTC was investigated in ten human tumour cell lines representing different mechanisms of cytotoxic drug resistance using the fluorometric microculture cytotoxicity assay (FMCA). In the cell line panel, L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine (P2) was the most active oligopeptide, showing slightly lower mean IC50 values (2.6 vs 3.9 and 4.1 microg ml(-1)) than Mel and m-L-SL. The other five oligopeptides were less active than Mel. All active oligopeptides showed mechanistic similarity to Mel as judged by the correlation analysis of the cell line panel log IC50 values (R > or = 0.90), although P2 appeared to be less sensitive to GSH-mediated drug resistance. The relative activity of Mel and P2 was found to be related to degree of proliferation, P2 being more active towards low-proliferating cell lines. P2 and Mel were then further characterized in 49 fresh human tumour samples. In these samples P2 was considerably more active than Mel and showed a higher relative solid tumour activity (2.7 to 4.5-fold). However, the correlation of log IC50s between P2 and Mel in patient cells was high (R = 0.79), indicating a similar mechanism of action in this tumour model too. Cross-resistance with other standard drugs was lower for P2 than Mel. The results show that P2 is the most potent component of PTC and demonstrates a favourable activity profile compared with Mel. These data suggest that further investigation of P2 as a potential anti-tumour agent is warranted.

Cell Line↗
PubMed1998

RBE for the induction of apoptosis in human peripheral lymphocytes exposed in vitro to high-LET radiation generated by accelerated nitrogen ions.

PURPOSE: To investigate the relative biological effectiveness (RBE) of accelerated nitrogen ions (32-45 MeV/u) compared with 137Cs gamma-rays for the induction of apoptosis in G0 lymphocytes. MATERIALS AND METHODS: Human peripheral G0 lymphocytes were exposed in vitro to doses up to 3 Gy. RBE for the induction of apoptosis was studied at different times after irradiation (0, 24, 48 and 72 h) with the use of three different methods: (1) morphological characterization of apoptotic cells after fluorescence staining; (2) cell size distribution analysis of the cell population to detect apoptotic bodies; and (3) electrophoretic analysis of DNA to detect 'DNA-ladders'. RESULTS: RBE values in the range of 1.3-3.0 were obtained from the linear components of the dose response curves. The variation in RBE was primarily dependent on post-irradiation time, where the highest RBE values were obtained after 48 h. The three different techniques used for analysis of apoptosis gave similar results. The significantly increased RBE was also seen as an earlier appearance of DNA-ladders, as well as a more rapid disappearance of the total number of viable cells. CONCLUSIONS: These results show that nitrogen ions of high linear energy transfer (LET) produce RBE > 1.0 and induce a faster apoptotic response as compared with gamma-photons of low-LET radiation.

Apoptosis↗
PubMed1998

Phase III trial of modulation of cisplatin/fluorouracil chemotherapy by interferon alfa-2b in patients with recurrent or metastatic head and neck cancer. Head and Neck Interferon Cooperative Study Group.

PURPOSE: In preclinical experiments, interferon alfa modulates the anticancer activity of fluorouracil (5-FU) and cisplatin (CDDP). To test this effect clinically in patients with recurrent or metastatic head and neck cancer (RMHNC), a multicenter randomized controlled trial with CDDP and 5-FU with or without interferon alfa-2b (IFNalpha) was performed. PATIENTS AND METHODS: Eligible patients had histologically confirmed RMHNC; a good performance status; measurable disease; adequate bone marrow, hepatic, and renal function; no prior chemotherapy for recurrent or metastatic disease; only one chemotherapy regimen administered with previous local therapy; and a treatment-free interval of at least 3 months following previous local therapy. Patients were randomized and stratified according to treatment center, and prior radiotherapy and chemotherapy. The treatment regimen consisted of CDDP 100 mg/m2 on day 1 and 5-FU 1,000 mg/m2/d by continuous infusion for 96 hours (days 1 to 4), without (arm A) or with (arm B) IFNg alpha 3 x 10(6) U/d subcutaneously on days 1 to 5. Cycles were repeated every 21 days. RESULTS: One hundred twenty-two patients were entered on each arm. The response rate (RR) was similar in both arms (arm A: complete response [CR] 10.7%, partial response [PR] 36.4%; arm B: CR 6.8%, PR 31.6%) (.70 < P < .50). There was no difference in median survival between the two arms (arm A 6.3 months v arm B 6.0 months; P = .49). Anorexia, fever, leukopenia, and thrombocytopenia grade III to IV were significantly more frequent in the IFNalpha arm. CONCLUSION: Modulation of CDDP and 5-FU with IFNalpha as used in this study does not improve the RR or the median survival in patients with RMHNC. Patients on both study arms had a poor prognosis, which indicates the need for novel therapies.

Adult↗
PubMed1997

Hypothyroidism after external radiotherapy for head and neck cancer.

PURPOSE: To study the development of thyroid hypofunction in patients with head and neck cancers admitted for external radiotherapy. METHODS AND MATERIALS: Between November 1990 and July 1996, thyroid function was measured in 264 consecutive patients, where the entire thyroid gland or part of it was included in the target volume. The time to development of hypothyroidism (HT) was calculated from the start of the radiotherapy. RESULTS: The median follow-up period was 19 months. Seventeen patients (6%) developed elevated serum thyroid-stimulating hormone levels with depressed (free) thyroxine levels (i.e., clinical HT). Elevated serum thyroid-stimulating hormone level with normal (free) thyroxine levels (i.e., chemical HT) developed in 57 (22%). The median time to clinical HT was 15 months (range: 7 to 32). The median time to chemical HT was also 15 months (range: 2 to 28). The actuarial risk of developing clinical or chemical HT 3 years after treatment was 15 and 40%, respectively. The incidence of chemical HT was significantly higher (p = 0.041) when the whole thyroid was included in the target volume compared to patients where only part of the thyroid was irradiated. The same trend was seen as regards clinical HT (p = 0.063). For those 20 patients who underwent laryngectomy, there was an increased risk of both chemical and clinical HT (p = 0.011 and 0.019, respectively). Increasing age was associated with an increased risk of chemical HT (p = 0.001), but not of clinical HT (p = 0.553). Sex, tumor site, radiation dose, and combination of radiotherapy and chemotherapy were not significant factors for thyroid hypofunction. CONCLUSION: Depressed thyroid function is common after external radiotherapy for cancers of the head and neck. Routine testing for possible thyroid hypofunction should be included in the follow-up procedures, even many years after end of radiotherapy.

Actuarial Analysis↗
PubMed1997

Response to radiotherapy of human uterine cervix carcinoma is not correlated with rearrangements of the Ha-ras-1 and/or c-myc genes.

An association between the presence of the activated form of Ha-ras-1 and c-myc genes and increased cellular radioresistance has been shown in several cell lines. The aim of this study was to determine whether such an association could be observed in clinical tumour biopsies. We examined 70 tumour specimens and 51 samples of peripheral blood obtained from untreated patients with carcinoma of the uterine cervix mainly stage II and III. In addition to initial clinical tumour response to radiotherapy, radiosensitivity was also analysed by the subrenal capsule assay (SRCA). Mutations in exons 1 and 2 of the Ha-ras-1 gene were examined using PCR single-strand conformation polymorphism (PCR-SSCP) and direct sequencing; and restriction fragment length polymorphism of the Ha-ras-1 gene was analysed using Southern hybridisation. Eight (11%) out of 70 tumours contained mutations in exons 1 and 2 of the Ha-ras-1 gene. Eleven (22%) out of the 51 tumours displayed rearrangements of the Ha-ras-1 gene (six tumours (12%) showed alterations of allele length, one amplification and four (8%) loss of one Ha-ras-1 allele). In addition, 12 (17%) out of 70 patients demonstrated the presence of rare alleles. Only one of the 70 tumours contained an amplified c-myc gene. There was no significant correlation between either rearrangements of the structure of the Ha-ras-1 and/or c-myc genes or presence of rare alleles in tumours and tumour response to radiotherapy.

Alleles↗
PubMed1997

Kinetics of G1/S and G2/M transition in X-irradiated ataxia-telangiectasia cells.

Cell cycle transition defects of homozygous ataxia-telangiectasia (A-T) cells were studied by using a cell cycle flow calculation method, which evaluates the dynamics of cell cycle traverse. We compared five human lymphoblastoid cell lines (LCLs) from A-T homozygotes belonging to complementation group A (ARO, BRO, RJO) and group C (CSA, BMA) with three cell lines from healthy volunteers (KK-B2, MTB, HGL). The A-T cell lines ARO and BRO were derived from the same family. Cell growth and cell cycle traverse were followed for 72 h after X-irradiation with 1-6 Gy. LCLs from healthy volunteers immediately arrested in G1 in a dose-dependent pattern, while the A-T cells did not arrest in G1 until after 12 to 24 h. The time for the appearance of the G1 arrest of these cells was independent of complementation group. The delayed G1 arrest seen in the A-T cells paralleled a lack of induction of p53, as described by others. In respect to G2 arrest, A-T cells from complementation group C (CSA, BMA) arrested to the same extent as cells from healthy volunteers. On the other hand, the other LCLs from complementation group A arrested normally, while cells from ARO and BRO did not arrest in G2. The lack of G2 arrest in BRO cells was accompanied by unchanged cdc2p34 activity. In summary, a defective radiation-induced G1 arrest seems to be present in both complementation groups of A-T homozygotes, whereas a defective G2 arrest in not always observed. The defective G1 arrest seen in A-T cells may play an important role in tumor cell survival after exposure to therapeutic irradiation.

Ataxia Telangiectasia↗
PubMed1996

Pharmacokinetics of cisplatin and its monohydrated complex in humans.

The pharmacokinetics of cisplatin and its cytotoxic hydrolysis product cis-diammineaquachloroplatinum(II) ion (monohydrated complex) were investigated in seven patients after they received a 1-h infusion of cisplatin in normal saline at 100 mg/m2. The concentrations of intact cisplatin and the monohydrated complex were determined in blood by liquid chromatography with post-column derivatization, using diethyldithiocarbamate as the reagent. A pharmacokinetic model was developed assuming that a fraction of the dose (2.3%) is present as the monohydrated complex in the infusion solution and that reversible reactions between cisplatin and its monohydrated complex prevail. The clearances of cisplatin and the monohydrated complex were 0.32 +/- 0.05 and 0.27 +/- 0.11 L/min/m2, respectively. The apparent volume of distribution was considerably smaller for the monohydrated complex (4 +/- 2 L/m2) than for cisplatin (11 +/- 2 L/m2). The elimination rate constants were 0.030 +/- 0.002 and 0.07 +/- 0.02 min-1 for cisplatin and the monohydrated complex, respectively. The area under the time-concentration curve for the monohydrated complex was approximately 15% of that for cisplatin. It is concluded that the significant amounts of the monohydrated complex present in blood are due to the fraction already present in the administered dose and to the fraction formed in blood.

Antineoplastic Agents↗