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Biomedical subjects

R Lewensohn

Publications and source records attributed to R Lewensohn.

At least 19 recordsLinked to original sources
PubMed2006

Ionizing radiation activates IGF-1R triggering a cytoprotective signaling by interfering with Ku-DNA binding and by modulating Ku86 expression via a p38 kinase-dependent mechanism.

Ionizing radiation exposure results in the activation of several tyrosine kinase receptors that participate in radiation-induced DNA damage response and radioresistance. We previously showed that insulin-like growth factor 1 receptor (IGF-1R) inhibition enhanced radiosensitivity of non-small-cell lung cancer (NSCLC) cells. In this paper, we demonstrate that in U1810 NSCLC cells gamma-radiation activates IGF-1R within 10 min, with a maximal activation effect 2 h post-irradiation. Impairment of IGF-1R tyrosine kinase activity enhances human lung cancer cells radiosensitivity by a mechanism that involves phosphatidylinositol 3-kinase (PI3-K) and p38 kinase. In an active form, IGF-1R binds and activates p38 kinase, promoting receptor signaling. Conversely, inhibition of IGF-1R phosphorylation results in IGF-1R/p38 complex disruption and p38 kinase inactivation. We have also demonstrated that in insulin-like growth factor-1-stimulated cells, Ku-DNA-binding activation is induced by ionizing radiation within 4 h, reaches a maximum level at 12 h and remains active up to 72 h. Blockade of IGF-1R activity or its downstream signaling through p38 kinase induces a decrease in radiation-mediated Ku-DNA-binding activation and downregulates the level of Ku86, without affecting Ku70 expression in the nucleus of U1810 cells. The IGF-1R signaling via PI3-K does not interfere with the p38 signaling, the Ku-DNA-binding activity or the level of Ku86. Our present study demonstrates for the first time that ionizing radiation activates IGF-1R. Inhibition of IGF-1R signaling via p38 kinase induces radiosensitivity by a novel mechanism involving nuclear Ku86.

Antigens, Nuclear↗
PubMed2006

Expression of DNA damage response proteins and complete remission after radiotherapy of stage IB-IIA of cervical cancer.

The primary aim of this study was to investigate if the expression of the DNA damage identifying protein DNA-PKcs known to be involved in DNA repair after treatment with ionising radiation can be used as a predictive marker for radiotherapy (RT) response in cervical cancer. Formalin-fixed primary tumour biopsies from 109 patients with cervical cancer, FIGO-stage IB-IIA, treated with preoperative brachytherapy followed by radical surgery were analysed by immunohistochemistry. In addition, correlation studies between early pathological tumour response to radiation and expression of Ku86, Ku70, Mdm-2, p53 and p21 in primary tumours were also performed. We found that tumour-transformed tissue shows positive immunostaining of DNA-PKcs, Ku86 and Ku70, while non-neoplastic squamous epithelium and tumour-free cervix glands show negative immunoreactivity. Expression of DNA-PKcs positively correlated with both Ku86 and Ku70, and a statistically significant correlation between the Ku subunits was also found. After RT, 85 patients demonstrated pathologic complete remission (pCR), whereas 24 patients had residual tumour in the surgical specimen (non-pCR). The main finding of our study is that there was no correlation between the outcome of RT and the expression of DNA-PK subunits. Positive p53 tumours were significantly more common among non-pCR cases than in patients with pCR (P=0.031). Expression of p21 and Mdm-2 did not correlate with the outcome of RT.

DNA Damage↗
PubMed2003

Low and high LET radiation-induced apoptosis in M059J and M059K cells.

PURPOSE: To investigate and compare the ability of DNA-dependent protein kinase (DNA-PK)-deficient and -proficient cells to undergo apoptosis after exposure to low and high linear energy transfer (LET) radiation. MATERIALS AND METHODS: A human glioma cell line M059J lacking the catalytic subunit of DNA-PK (DNA-PKcs) and its DNA-PKcs-proficient counterpart, M059K, were exposed to 1 and 4 Gy of accelerated nitrogen ions (14N, 140 eV nm(-1), 8-12 Gy min(-1)) or 60Co gamma-rays (0.2 eV nm(-1), 0.7 Gy min(-1)). The induction of apoptosis was studied up to 144 h post-irradiation using two different methods: morphological characterization of apoptotic cells after fluorescent staining and cell size distribution analysis to detect apoptotic bodies. In parallel, protein expression of DNA-PKcs and poly(ADP-ribose) polymerase (PARP) as well as DNA-PK and caspase-3 activity were investigated. RESULTS: Low and high LET radiations (4 Gy) induced a time-dependent apoptotic response in both cell lines. Low LET radiation induced a significantly elevated apoptotic response in M059J as compared with M059K cells at 144 h post-irradiation. Following high LET radiation exposure, there was no difference between the cell lines at this time. PARP cleavage was detected in M059J cells following both low and high LET irradiation, while only high LET radiation induced PARP cleavage in M059K cells. These cleavages occurred in the absence of caspase-3 activation. CONCLUSIONS: M059J and M059K cells both display radiation-induced apoptosis, which occur independently of caspase-3 activation. The apoptotic course differs between the two cell lines and is dependent on the quality of radiation.

Apoptosis↗
PubMed2003

Difference in the induction, but not in the repair, of X-ray- and nitrogen ion-induced DNA single-strand breaks as measured using human cell extracts.

PURPOSE: To compare the repair efficiency of X-ray (low linear energy transfer [LET]) and nitrogen ion (high LET)-induced single-strand breaks (SSB) in a human cell-free end-joining system. MATERIALS AND METHODS: SSB were introduced into a bacterial plasmid, pBR322, by X-rays (4 MeV photons) and nitrogen ions with an LET=125 keV micro m(-1). Repair efficiency was studied under incubation with the protein extracts from human squamous carcinoma cells, UT-SCC-5. RESULTS: A several fold higher dose of nitrogen ion radiation compared with X-ray radiation was needed to induce a similar loss of supercoiled plasmid DNA. There was no difference in the repair efficiency of SSB induced by these two types of radiation. CONCLUSION: The data indicate that X-rays at 25 Gy and nitroging ions at 100 Gy radiation doses, under condition of low scavenging capacity (10 mM Tris), induce SSB of similar complexity or, alternatively, differences in SSB complexity do not alter the repair rate.

Carcinoma, Squamous Cell↗
PubMed2001

Defective caspase-3 relocalization in non-small cell lung carcinoma.

Many anticancer drugs exert their cytotoxicity through DNA damage and induction of apoptosis. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) have different sensitivity to treatment with radiation and chemotherapeutic agents with SCLC being more sensitive than NSCLC both in vitro and in vivo. This difference might be related to the different susceptibility of small and non-small cell lung carcinoma to undergo apoptosis. The aim of this study was to investigate if deficiencies in the apoptotic pathways can explain the intrinsic resistance of NSCLC to anti-cancer treatment. Three different triggers were used to induce apoptosis. Etoposide and gamma-radiation, which are important parts of clinical lung cancer treatment, induce DNA-damage, whereas Fas ligation induces receptor-mediated apoptotic pathways. NSCLC cells were cross-resistant to all treatments, whereas SCLC cells, which do not express pro-caspase-8, were resistant to alphaFas-, but not to DNA-damage-induced apoptosis. Cytochrome c release, activation of caspase-9 and the executioner caspase-3 were observed in both types of lung cancer cells. However, cleavage of known nuclear substrates for caspase-3, such as PARP and DFF45/ICAD, was documented only in the sensitive SCLC cells but not in the resistant NSCLC cells. Moreover, relocalization of active caspase-3 from the cytosol into the nucleus upon treatment was observed only in the SCLC cell line. These results indicate that the inhibition of apoptosis in NSCLC occurs downstream of mitochondrial changes and caspase activation, and upstream of nuclear events.

Apoptosis↗
PubMed2001

DNA-dependent protein kinase is inhibited by trifluoperazine.

The DNA-dependent protein kinase (DNA-PK) is a serine/threonine nuclear kinase, important for the repair of DNA double strand breaks (DSB). Cells defective in DNA-PK show increased sensitivity to ionising radiation and different DNA-damaging drugs, such as cisplatinum. Increased sensitivity to cisplatinum has previously been noted in the presence of phenothiazines. We tested a panel of phenothiazines and one thioxanthen for any influence upon the activity and expression of DNA-PK in a nonsmall cell lung cancer cell line, U-1810. The activity of DNA-PK was completely inhibited in cell lysate and in purified enzyme by 200 microM TFP. DNA-PKcs and Ku86 cleavage were evident in U-1810 cells after 30 min incubation with 100 microM TFP, along with changes in the cells consistent with apoptosis. Our study suggests that phenothiazines and thioxanthens, acting through DNA-PK, have the potential to enhance the effects of DNA damaging agents.

Blotting, Western↗
PubMed2001

Radiosensitivity of human squamous carcinoma cell lines is associated with amount of spontaneous DNA strand breaks.

We asked whether the constitutive level of DNA strand breaks (SBs) in four human squamous carcinoma cell lines is associated with their radiosensitivity, measured by the clonogenic assay. Because impairment in DNA replication and the action of endogenous deoxyribonucleases are two major sources of DNA strand breaks under normal cell metabolism, we also analyzed DNA polymerase and DNA ligase activities as well as the functional status of Poly(ADP-ribose) polymerase (PARP) and nucleolytic degradation of genomic DNA. We showed that the two relatively radioresistant cell lines, UM-SCC-1 and UT-SCC-5, had a statistically significant lower constitutive level of DNA SBs, as measured by DNA precipitation technique, compared with the two relatively radiosensitive cell lines, UM-SCC-14A and UT-SCC-9. We found that cell lines with a higher level of broken DNA tended to have a higher constitutive level of DNA polymerase alpha activity, measured by incorporation of [(3)H]dTTP in DNase I-activated DNA. UM-SCC-1, UT-SCC-5, and UM-SCC-14A did not show any difference in DNA ligase activity when a nicked oligonucleotide was used as substrate. The most radiosensitive cell line, UT-SCC-9, had a significantly lower ligation efficiency compared to the other three cell lines. The functional status of the PARP was the same in the four cell lines. Although none of the four cell lines showed a characteristic apoptotic or necrotic degradation of genomic DNA, when tested with the "plasmid rejoining assay," a significant degradation of the plasmid DNA in UT-SCC-9 was detected. We conclude that the high fraction of DNA SBs for UT-SCC-9, the most radiosensitive cell line, is most likely a consequence of low ligation efficiency combined with a relatively high DNA polymerase alpha activity and the nuclease degradation of DNA.

Apoptosis↗
PubMed2001

A Swedish study of chemoradiation in squamous cell carcinoma of the esophagus.

This multicenter study describes the development of a chemoradiation protocol for the treatment of non-metastatic squamous cell carcinoma of the esophagus. Eighty patients were treated with three courses of chemotherapy (cisplatinum and 5-fluorouracil) with concomitant radiotherapy (40 Gy) during the last two courses of chemotherapy. Esophagectomy was performed, when feasible. If no operation was performed, patients were planned to receive a target dose of 64 Gy. Toxicity was mainly attributable to hematological impairment and led to two adjustments of the treatment protocol (addition of filgrastim and lowering of the 5-fluorouracil dose). These changes made it possible to administer the planned treatment in a gradually higher proportion of patients (13/23 [57%] before changes of treatment compared with 30/36 [83%] after changes). Treatment-related mortality was 3.75% (3 patients, associated with leucopenic septicemia after chemotherapy). Fifty-four patients were resected. No per- or postoperative mortality was encountered. The complete response (pathological CR) rate in operated patients was 46% (27/59 patients) after chemoradiation. In the whole series the CR rate (including clinical CR for non-resected patients) was 44%. With a minimum follow-up of 37 months, the 3-year survival for the whole group was 31% compared with 57% for the CR patients. Total 5-year survival thus far (July 1999) is 26%.

Adult↗
PubMed2001

High-LET radiation induces apoptosis in lymphoblastoid cell lines derived from atazia-telangiectasia patients.

PURPOSE: To investigate and compare the propensity of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL), derived from ataxia-telangiectasia (A-T) patients and from unaffected healthy individuals (controls), to undergo apoptosis after exposure to high-linear energy transfer (LET) radiation. MATERIALS AND METHODS: Four A-T (ARO, BMA, CSA and RJO) and two control (JAC and KKB3) LCL were exposed to doses of up to 4Gy of accelerated nitrogen ions (32-45 MeV/u, 8-12Gy/min). For comparative purposes X-ray irradiation (1.36 Gy/min) was also performed. The induction of apoptosis was studied 0-48 h after irradiation with the use of two methods: (1) monitoring of high molecular weight (HMW) DNA fragments by field inversion pulse gel electrophoresis (FIGE); and (2) morphological characterization ofapoptotic cells after fluorescent staining. In parallel, cell-cycle distribution, monitored by DNA flow cytometry, as well as measurements of p53/p21(WAF1) protein levels by Western blots, were investigated in these cells. RESULTS: High-LET radiation-induced apoptosis and G2/M-arrest in both A-T and control LCL. No significant increase in the amount of p53/p21(WAF1) proteins preceded apoptosis in control or in A-T LCL after high-LET irradiation. However, low-LET radiation did induce significant enhanced levels of p53 proteins in control but not in A-T LCL. CONCLUSIONS: LCL from both A-T homozygous and unaffected healthy individuals undergo apoptosis without accumulation of p53/p21(WAF1) proteins after exposure to high-LET radiation. In contrast, low-LET radiation induces apoptosis and significantly increases levels of p53 protein in control but not in A-T LCL.

Apoptosis↗
PubMed2001

Cellular effects of high-intensity focused continuous wave ultrasound alone and in combination with X-rays.

PURPOSE: To investigate the cellular effects of 1.1 MHz high-intensity focused continuous wave (CW) ultrasound alone and in combination with X-rays. MATERIALS AND METHODS: V79 cells were exposed to ultrasound of different intensities for 30s (non-hyperthermic conditions). In the combined treatments, cells were exposed to ultrasound (63 W/cm2) either before or after 2 Gy X-irradiation. Cellular effects studied were clonogenic survival, DNA strand breaks (pulsed-field gel electrophoresis, DNA precipitation assay) and membrane integrity (morphological analysis). RESULTS: With increasing ultrasound intensity, cell survival decreased in a dose-dependent manner as the induction of DNA strand breaks, the fraction of cells with lost membrane integrity and cell lysis increased. In the treatments with combined exposures, the regimen with X-rays before ultrasound had a nearly additive effect on cell kill, whereas the reverse regimen with ultrasound exposure before X-irradiation resulted in a synergistic effect (p<0.012). CONCLUSIONS: High-intensity focused CW ultrasound induces an intensity-dependent reduction in clonogenic survival in V79 cells, which seems to depend on both DNA and membrane damage. Combined exposures of ultrasound and X-rays resulted in a synergistic reduction in cell survival when cells were exposed to ultrasound before X-rays but not for the reverse regimen. Thus, a larger fraction of the repairable sublethal cell damage induced by an initial ultrasound exposure was rendered non-repairable by a subsequent X-ray exposure than if the reverse treatment order was used.

Animals↗
PubMed2000

Frequent amplification of the telomerase reverse transcriptase gene in human tumors.

Activation of telomerase is a crucial step during cellular immortalization and malignant transformation of human cells and requires the induction of the catalytic component, human telomerase reverse transcriptase (hTERT), encoded by the hTERT gene. It is poorly understood how the hTERT gene is activated in human cancer cells. In the present study, we examined the hTERT gene copy number in human cancer cell lines and in primary tumor tissues. Amplification of the hTERT gene was observed in 8 of 26 (31%) tumor cell lines and 17 of 58 (30%) primary tumors examined (8 of 21 lung tumors, 3 of 10 cervical tumors, 5 of 19 breast carcinomas, and 1 of 8 neuroblastomas). In addition, 13 of 26 (50%) cell lines and 13 of 58 (22%) primary tumors displayed gain of hTERT gene copies with 3-4 copies/cell. The present findings imply that the hTERT locus may be a frequent target for amplification during tumorigenesis and that this genetic event probably contributes to the dysregulation of telomerase activity occurring in human tumors.

Catalytic Domain↗
PubMed2000

Human papillomavirus (HPV) DNA in tonsillar cancer: clinical correlates, risk of relapse, and survival.

Human papillomavirus (HPV) is more commonly found in tonsillar cancer than in other head and neck cancers. The importance of HPV status in tonsillar cancer for prognosis remains unclear. The aim of the present study was to investigate the frequency of HPV in tonsillar cancer and to correlate the presence of HPV with tumor stage, nodal status, grade of differentiation, risk of relapse, and survival. HPV DNA and HPV type were determined, using PCR, in pre-treatment biopsies from 60 cases of primary tonsillar cancer. All patients had undergone full-dose radiotherapy, 45% as the only treatment modality, and 55% in combination with surgery. HPV 16 was detected in 43% (26/60) of the cancers including 1 double infection of both HPV 16 and HPV 33. Patients with HPV(+) tonsillar cancer showed less risk of relapse within 3 years after diagnosis, with a better odds ratio of 4.18 as compared with HPV(-) patients (p = 0. 025). Furthermore, cause specific survival was significantly (p = 0. 047) better in patients with HPV(+) tonsillar carcinomas. At 3 years after diagnosis the survival rate was 65.3% in the HPV(+) group and 31.5% in the HPV(-) group, and at 5 years the survival rate was 53. 5% and 31.5%, respectively. The better outcome for patients with HPV(+) tonsillar cancer was independent of TNM stage, nodal status, gender and age. These results indicate that HPV status is a significantly favorable prognostic factor in tonsillar cancer and may be used as a marker in order to optimize the treatment of patients with this type of cancer.

Adult↗
PubMed2000

[Radioimmunotherapy is rapidly developing to clinically efficient therapy].

The use of monoclonal antibodies (mabs) in cancer therapy has gained renewed interest, due to recent reports of remarkable clinical response, particularly in patients with low-grade non-Hodgkin lymphoma. Better defined and more appropriate target antigens and "humanized" mabs, reducing the risk of inducing neutralising human anti-mouse antibodies, have contributed to the improvement in results. Conjugation of mabs with various radionuclides is now being explored as a means of further enhancing clinical efficacy, the idea being to allow systemic delivery of targeted radiation to areas of disease while sparing normal tissue. Radioimmunotherapy may be administered as a single large dose of radiolabelled mabs, usually requiring haematological stem cell support, or as multiple, smaller fractions. The criteria for the selection of mabs and radionuclides are discussed in the article, as are recent clinical data and the problems and prospects of future developments in radioimmunotherapy.

Clinical Trials as Topic↗
PubMed2000

Role of apoptosis in the response of lung carcinomas to anti-cancer treatment.

Resistance of tumor cells to treatment often accounts for the failure of traditional forms of anti-cancer therapy. It is well known that tumors from the same histological group and stage of development are highly heterogeneous in their sensitivity to therapy. Among the factors that can influence tumor sensitivity are DNA repair capacity, distribution of cells throughout the cell cycle, proliferation potential, etc. In many cases, anti-cancer therapy eliminates tumor cells via apoptosis, an active form of cell death characterized by cell shrinkage and the removal of cells in a neat, orderly fashion. However, this process is not always efficient. In the present review, the precise role that apoptosis plays in the response of lung carcinomas to chemotherapy and radiation treatment is discussed.

Animals↗
PubMed1999

Prognostic impact of complete remission after preoperative irradiation of tonsillar carcinoma: a retrospective analysis of the radiumhemmet data, 1980-1995.

PURPOSE: This retrospective study was done to determine the outcome of patients with tonsillar carcinoma treated at Radiumhemmet, Karolinska Hospital, between January 1980 and December 1995 with radiotherapy alone or in combination with surgery. In addition the importance of tumor remission for patient survival was analyzed. METHODS AND MATERIALS: The analysis is based on 167 previously untreated patients with biopsy-proven, invasive tonsillar squamous cell carcinoma of the tonsillar region. All patients were consecutively admitted to the Department of General Oncology, Radiumhemmet, and treated with curative intent. The median follow-up time was 20 months. The median target dose was 64 Gy, delivered in fractions of 2 Gy 5 times weekly. Twenty-eight percent of the patients underwent surgery of the primary site and/or neck dissection after radiotherapy (RT). RESULTS: The overall local control rate for the whole patient group after radiotherapy was 79%. Probability of survival after 5 years for patients responding with complete remission (CR) was 43% and for patients with incomplete response (non-CR) 9%, (p<0.0001). The survival in the non-CR group treated with combination therapy was 20 months longer than in patients receiving radiotherapy alone (p<0.0001). There was no statistically significant difference in prediction of long-term survival when the patient population was stratified according to tumor differentiation grade, age, sex, nodal status, or treatment time. CONCLUSION: The strongest clinical predictor of survival was the degree of tumor remission after RT. For the non-CR group receiving combination treatment including surgery there was a survival benefit as compared to patients treated with RT alone (p<0.0001) although there were few long-term survivors in this patient group.

Carcinoma, Squamous Cell↗
PubMed1999

The immunohistochemical expression of DNA-PKCS and Ku (p70/p80) in head and neck cancers: relationships with radiosensitivity.

PURPOSE: The DNA-PK complex is one of the major pathways by which mammalian cells respond to DNA double-strand breaks induced by ionizing radiation. This study evaluated the relationship between the immunohistochemical expression of the individual components of DNA-PK and cellular radiosensitivity in head and neck cancers. METHODS AND MATERIALS: Biopsies from patients with previously untreated squamous cell carcinomas of the head and neck were assessed for inherent tumor radiosensitivity measured as the surviving fraction at 2 Gy (SF2) using a soft agar clonogenic assay. Paraffin-embedded tumor material from 64 successfully grown specimens was immunohistochemically stained for expression of DNA-PKcs and Ku (p70/p80). The same tumor material was previously analyzed for the immunohistochemical expression of p53. RESULTS: A significant correlation was found between the degree of expression of DNA-PKcs and Ku (p70/p80) (r = 0.55, p<0.001). There were no overall significant differences in the levels of expression of DNA-PKcs and Ku (p70/p80) in tumors from patients of either sex, different sites, histologies, and stages. No relationship was found between SF2 and the expression of either DNA-PKcs (r = 0.22, p = 0.081) or Ku (p70/p80) (r = 0.064, p = 0.62). Comparison with previous immunohistochemical characterization showed no significant correlations between the expression levels of p53 and either DNA-PKcs (r = 0.093, p = 0.46) or Ku (p70/p80) (r = -0.17, p = 0.17). CONCLUSIONS: This study suggests that determining the immunohistochemical expression of DNA-PK in head and neck cancers from multiple sites does not have a role as a predictive assay of tumor in vitro radiosensitivity.

Antigens, Nuclear↗
PubMed1999

Differences in expression of pro-caspases in small cell and non-small cell lung carcinoma.

Expression of several molecular determinants of apoptosis was analyzed in 10 untreated small cell (SCLC) and 6 untreated non-small cell (NSCLC) lung carcinoma cell lines. Although SCLC lines were more prone to spontaneous apoptosis compared with NSCLC lines, the former showed higher Bcl-2 expression and a higher Bcl-2/Bax ratio. In order to understand this apparent contradiction, the expression of pro-caspases as well as calpain was analyzed in these cell lines at the protein and mRNA levels. No differences in protein level of pro-caspases-2, -3, -7, and -9 and of calpain were detected between the SCLC and the NSCLC lines, but a striking difference in pro-caspase-8 expression was noted. All 6 NSCLC, but only 2 of the 10 SCLC lines, expressed pro-caspase-8 protein. Further experiments using the RNase protection assay indicated that the lack of pro-caspase-8 expression at the mRNA level was characteristic for SCLC. Using the same experimental approach, we found that SCLC cell lines in addition to pro-caspase-8 were deficient in mRNA expression of pro-caspases-1, -4, and -10, suggesting a different caspase-activating cascade in SCLC compared with NSCLC. This first systematic characterization of pro-caspase expression in lung cancer surprisingly showed that SCLC, which are more prone to undergo spontaneous apoptosis, are deficient in several pro-caspases and have a high Bcl-2/Bax ratio. Thus, the propensity of SCLC cells to undergo apoptosis cannot be explained only by the expression of factors involved in regulation or execution of apoptosis.

Apoptosis↗