The Effects of Proteasome Inhibitors on Telomerase Activity and Regulation in Multiple Myeloma Cells.

The importance of telomerase, the enzyme that maintains telomere length, has been reported in many malignancies in general and in multiple myeloma (MM) in particular. Proteasome inhibitors are clinically used to combat effectively MM. Since the mechanism of action of proteasome inhibitors has not been fully described we sought to clarify its potential effect on telomerase activity (TA) in MM cells. Previously we showed that the first generation proteasome inhibitor bortezomib (Brt) inhibits TA in MM cells by both transcriptional and post-translational mechanisms and has a potential clinical significance. In the current study we focused around the anti- telomerase activity of the new generation of proteasome inhibitors, epoxomicin (EP) and MG-132 in order to clarify whether telomerase inhibition represents a class effect. We have exposed MM cell lines, ARP-1, CAG, RPMI 8226 and U266 to EP or MG and the following parameters were assessed: viability; TA, hTERT expression, the binding of hTERT (human telomerase reverse transcriptase) transcription factors and post-translational modifications. Epoxomicin and MG-132 differentially downregulated the proliferation and TA in all MM cell lines. The downregulation of TA and the expression of hTERT were faster in CAG than in ARP-1 cells. Epoxomicin was more potent than MG-132 and therefore further mechanistic studies were performed using this compound. The inhibition of TA was mainly transcriptionally regulated. The binding of three positive regulator transcription factors: SP1, c-Myc and NF-κB to the hTERT promoter was decreased by EP in CAG cells as well as their total cellular expression. In ARP-1 cells the SP1 and c-MYC binding and protein levels were similarly affected by EP while NF-κB was not affected. Interestingly, the transcription factor WT-1 (Wilms' tumor-1) exhibited an increased binding to the hTERT promoter while its total cellular amount remained unchanged. Our results combined with our previous study of bortezomib define telomerase as a general target for proteasome inhibitors. The inhibitory effect of TA is exerted by several regulatory levels, transcriptional and post translational. SP1, C-Myc and NF-κB were involved in mediating these effects. A novel finding of this study is the role of WT-1 in the regulation of telomerase which appears as a negative regulator of hTERT expression. The results of this study may contribute to future development of telomerase inhibition as a therapeutic modality in MM.

Characterisation of blood-derived exosomal hTERT mRNA secretion in cancer patients: a potential pan-cancer marker.

BACKGROUND: Telomerase (human telomerase reverse transcriptase (hTERT)) is considered a hallmark of cancer. The aim of our study was to evaluate the feasibility of the detection of hTERT transcripts in serum as a 'pan-cancer' diagnostic method. METHODS: Human telomerase reverse transcriptase mRNA levels were determined in serum and serum-derived exosomes from 133 patients with different malignancies and 45 healthy controls. In four patients hTERT mRNA levels were measured in different clinical stages. RESULTS: Human telomerase reverse transcriptase transcript was absent in all controls and was variably detected in 67.5% of patients with all cancer types. A correlation between hTERT transcript levels and the clinical course was found in several cases. CONCLUSIONS: Human telomerase reverse transcriptase mRNA levels may reflect the tumour burden and the clinical status of the patient. In patients with detectable levels, this assay may potentially serve as a diagnostic and follow-up 'pan-cancer' marker. Owing to the large variety of patients and small sample size in each diagnosis, the statistical power is limited and will be explored further in larger groups.

[EXOSOMES AS MEDIATORS OF INTERCELLULAR COMMUNICATION: THE CANCER AND TELOMERASE CONNECTION].

INTRODUCTION: Exosomes, nano-vesicles secreted from all types of cells in the human body, function as inter-cell communicators. This role of exosomes is fulfilled by their specific content, dependent on the origin of donor cells from which they are secreted. Exosomes contain a plethora of nucleic acids (DNA, RNA and micro RNA), proteins and lipids. These molecules are packed in the donor cells into the exosomes which are subsequently secreted and transferred through various body fluids into the target cells which may be located far from the donor cells. Recently, the issue of exosome research has been widely expanded and data has been accumulated regarding the relevance and involvement of exosomes in cancer. Exosomes play a role in the process of malignant transformation, in avoiding the surveillance of the immune system, the expansion of the tumor and its establishment in the cancer microenvironment. In addition, exosomes promote the process of metastasis formation. Among other subjects, exosomal research focuses around the characterization of the content of cancer-derived exosomes in order to identify markers that can be used for diagnosis and prognosis of cancer patients. Telomerase, a unique reverse transcriptase, has been widely shown to be crucial for the process of malignant transformation and the perpetuation of the malignant clone. In the current review, we describe the importance of exosomes to the various themes of cancer and their potential use as diagnostic and prognostic markers as well as their therapeutic potential. In addition, the results and implications of our study with regards to the secretion of telomerase transcript into exosomes derived from cancer cells will be discussed.

The effects of erythropoietin signaling on telomerase regulation in non-erythroid malignant and non-malignant cells.

Treatment with erythropoietin (EPO) in several cancers is associated with decreased survival due to cancer progression. Due to the major importance of telomerase in cancer biology we hypothesized that some of these effects may be mediated through EPO effect on telomerase. For this aim we explored the possible effects of EPO on telomerase regulation, cell migration and chemosensitivity in non-erythroid malignant and non-malignant cells. Cell proliferation, telomerase activity (TA) and cell migration increased in response to EPO. EPO had no effect on cancer cells sensitivity to cisplatinum and on the cell cycle status. The inhibition of telomerase modestly repressed the proliferative effect of EPO. Telomere shortening caused by long term inhibition of the enzyme abolished the effect of EPO, suggesting that EPO effects on cancer cells are related to telomere dynamics. TA was correlated with the levels of Epo-R. The increase in TA was mediated post-translationally through the Lyn-Src and not the canonical JAK2 pathway.

Chronic Lymphocytic Leukemia (CLL)-Derived Extracellular Vesicles Educate Endothelial Cells to Become IL-6-Producing, CLL-Supportive Cells.

We hypothesized that via extracellular vesicles (EVs), chronic lymphocytic leukemia (CLL) cells turn endothelial cells into CLL-supportive cells. To test this, we treated vein-derived (HUVECs) and artery-derived (HAOECs) endothelial cells with EVs isolated from the peripheral blood of 45 treatment-naïve patients. Endothelial cells took up CLL-EVs in a dose- and time-dependent manner. To test whether CLL-EVs turn endothelial cells into IL-6-producing cells, we exposed them to CLL-EVs and found a 50% increase in IL-6 levels. Subsequently, we filtered out the endothelial cells and added CLL cells to this IL-6-enriched medium. After 15 min, STAT3 became phosphorylated, and there was a 40% decrease in apoptosis rate, indicating that IL-6 activated the STAT3-dependent anti-apoptotic pathway. Phospho-proteomics analysis of CLL-EV-exposed endothelial cells revealed 23 phospho-proteins that were upregulated, and network analysis unraveled the central role of phospho-ß-catenin. We transfected HUVECs with a ß-catenin-containing plasmid and found by ELISA a 30% increase in the levels of IL-6 in the culture medium. By chromatin immunoprecipitation assay, we observed an increased binding of three transcription factors to the IL-6 promoter. Importantly, patients with CLL possess significantly higher levels of peripheral blood IL-6 compared to normal individuals, suggesting that the inducers of endothelial IL-6 are the neoplastic EVs derived from the CLL cells versus those of healthy people. Taken together, we found that CLL cells communicate with endothelial cells through EVs that they release. Once they are taken up by endothelial cells, they turn them into IL-6-producing cells.

Differences in Telomere Length between Adolescent Females with Anorexia Nervosa Restricting Type and Anorexia Nervosa Binge-Purge Type.

Physiological and psychological distress may accelerate cellular aging, manifested by shortening of telomere length (TL). The present study focused on TL shortening in anorexia nervosa (AN), an illness combining physiological and psychological distress. For that purpose, we measured TL in 44 female adolescents with AN at admission to inpatient treatment, in a subset of 18 patients also at discharge, and in 22 controls. No differences in TL were found between patients with AN and controls. At admission, patients with AN-binge/purge type (AN-B/P; n = 18) showed shorter TL compared with patients with AN-restricting type (AN-R; n = 26). No change in TL was found from admission to discharge, despite an improvement in body mass index standard deviation score (BMI-SDS) following inpatient treatment. Older age was the only parameter assessed to be correlated with greater TL shortening. Several methodological changes have to be undertaken to better understand the putative association of shorter TL with B/P behaviors, including increasing the sample size and the assessment of the relevant pathological eating disorder (ED) and non-ED psychological correlates in the two AN subtypes.

Blood-Derived Exosomal hTERT mRNA in Patients with Lung Cancer: Characterization and Correlation with Response to Therapy.

Background: Telomerase (human telomerase reverse transcriptase (hTERT) is considered a hallmark of cancer, being active in cancer cells but repressed in human somatic cells. As such, it has the potential to serve as a valid cancer biomarker. Exosomal hTERT mRNA can be detected in the serum of patients with solid malignancies but not in healthy individuals. We sought to evaluate the feasibility of measuring serum exosomal hTERT transcripts levels in patients with lung cancer. Methods: A prospective analysis of exosomal hTERT mRNA levels was determined in serum-derived exosomes from 76 patients with stage III-IV lung cancer (11 SCLC and 65 NSCLC). An hTERT level above RQ = 1.2 was considered "detectable" according to a previous receiver operating characteristic curve (ROC) curve. Sequential measurements were obtained in 33 patients. Demographic and clinical data were collected retrospectively from patients' charts. Data on response to systemic therapy (chemotherapy, immunotherapy, and tyrosine kinase inhibitors) were collected by the treating physicians. Results: hTERT was detected in 53% (40/76) of patients with lung cancer (89% of SCLC and 46% of NSLCC). The mean hTERT levels were 3.7 in all 76 patients, 5.87 in SCLC patients, and 3.62 in NSCLC patients. In total, 25 of 43 patients with sequential measurements had detectable levels of hTERT. The sequential exosomal hTERT mRNA levels reflected the clinical course in 23 of them. Decreases in hTERT levels were detected in 17 and 5 patients with partial and complete response, respectively. Eleven patients with a progressive disease had an increase in the level of exosomal hTERT, and seven with stable disease presented increases in its exosomal levels. Another patient who progressed on the first line of treatment and had a partial response to the second line of treatment exhibited an increase in exosomal hTERT mRNA levels during the progression and a decrease during the response. Conclusions: Exosomal hTERT mRNA levels are elevated in over half of patients with lung cancer. The potential association between hTERT levels and response to therapy suggests its utility as a promising cancer biomarker for response to therapy. This issue should be further explored in future studies.

Is serum-derived exosomal hTERT transcript a marker of oncogenic activity in primary brain tumors? An exploratory study.

BACKGROUND: In order to proliferate indefinitely, all tumors require a telomere maintenance mechanism. The expression of human telomerase reverse transcriptase (hTERT) enables telomere maintenance and provides cancer cells with limitless replicative potential. As such, it may serve as an attractive biomarker for oncogenic activity. This study explored whether a liquid biopsy that analyses blood derived exosomal hTERT transcript (e-hTERT-trans) may serve as such a biomarker in gliomas and meningiomas when compared to healthy controls. METHODS: Exosomes were isolated from the pre-operative sera of patients' samples stored in the biobank of both Rabin and Sheba Medical Centers. The levels of e-hTERT-trans were measured in 81 healthy controls, 117 meningiomas, 17 low-grade gliomas, and 61 glioblastomas. Clinical parameters of the patients were collected retrospectively and compared to the levels of the e-hTERT-trans. RESULTS: The upper normal limit of controls e-hTERT-trans was 1.85 relative quantitation (RQ). The rate of detection increased with rising tumor grade and correlated with tumor recurrence in meningiomas: mean RQ without recurrence (2.17 ± 11.7) versus with recurrence (3.59 ± 4.42; p = 0.002). In glioblastomas, preoperative measurements correlated with tumor volume and with the disease course on serial sampling. CONCLUSIONS: We demonstrated for the first time that the expression of e-hTERT-trans transcript can be measured in the serum of primary brain tumors. This exosomal marker carries the potential to serve as a biomarker once used in conjunction with other clinical and radiological parameters. Future studies are required to investigate whether the sensitivity could be augmented and whether it can be implemented into routine patients care.

Exosomal telomerase transcripts reprogram the microRNA transcriptome profile of fibroblasts and partially contribute to CAF formation.

It is now well accepted that cancer cells change their microenvironment from normal to tumor-supportive state to provide sustained tumor growth, metastasis and drug resistance. These processes are partially carried out by exosomes, nano-sized vesicles secreted from cells, shuttled from donor to recipient cells containing a cargo of nucleic acids, proteins and lipids. By transferring biologically active molecules, cancer-derived exosomes may transform microenvironmental cells to become tumor supportive. Telomerase activity is regarded as a hallmark of cancer. We have recently shown that the transcript of human telomerase reverse transcriptase (hTERT), is packaged in cancer cells derived- exosomes. Following the engulfment of the hTERT transcript into fibroblasts, it is translated into a fully active enzyme [after assembly with its RNA component (hTERC) subunit]. Telomerase activity in the recipient, otherwise telomerase negative cells, provides them with a survival advantage. Here we show that exosomal telomerase might play a role in modifying normal fibroblasts into cancer associated fibroblasts (CAFs) by upregulating [Formula: see text]SMA and Vimentin, two CAF markers. We also show that telomerase activity changes the transcriptome of microRNA in these fibroblasts. By ectopically expressing microRNA 342, one of the top identified microRNAs, we show that it may mediate the proliferative phenotype that these cells acquire upon taking-up exosomal hTERT, providing them with a survival advantage.

Characterization of blood-derived exosomal hTERT mRNA as a biomarker for colon cancer and Lynch syndrome.

Background: Human telomerase reverse transcriptase (hTERT)- mRNA was shown to be elevated in exosomes derived from the sera of a variety of hematological and solid cancer patients. We aimed to evaluate its role as a diagnostic marker in patients with newly diagnosed colon cancer and in hereditary syndromes with predisposition to colon cancer. Methods: hTERT -mRNA levels were determined in serum-derived exosomes from 88 patients with colon cancer, 71 Lynch-syndrome carriers with unknown active malignancies and 50 healthy controls. Data, including demographics, background diseases, clinical data regarding tumor characteristics and genetic data, were retrieved data from medical files. Results: Patients with colon cancer had both higher exosomal hTERT mRNA levels and a higher proportion of patients with positive exosomal hTERT mRNA than controls (29.5% vs. 4%, respectively, P values < 0.001). Within the cancer group, patients with a metastatic disease had higher levels of telomerase mRNA than non-metastatic disease patients, and these levels correlated with CEA levels. Likewise, Lynch syndrome carriers had a higher proportion of positive exosomal hTERT mRNA than controls (21.1% vs. 4%, respectively, P value 0.008) but only a trend towards higher exosomal hTERT mRNA levels. Higher telomerase mRNA levels were not correlated with the mutated gene. Conclusions: Exosomal serum hTERT -mRNA levels are associated with metastatic colon cancer and were also demonstrated in a subset of Lynch syndrome carriers. Its significance as a biomarker for developing malignancy should be elucidated.

Ionizing radiation up-regulates telomerase activity in cancer cell lines by post-translational mechanism via ras/phosphatidylinositol 3-kinase/Akt pathway.

PURPOSE: Telomerase is considered currently as a hallmark of cancer, and its inhibition is expected to become an important anticancer modality. In contrast to abundant data concerning the effect of cytotoxic drugs on telomerase activity (TA), there is scant information on the effect of radiation on telomerase. The mechanism of telomerase regulation by irradiation has never been evaluated in detail. In the present study, we investigated the effect of radiation on TA and its regulation in cancer cells. EXPERIMENTAL DESIGN: The effect of various radiation doses on TA in several malignant and nonmalignant cell lines was evaluated. All malignant cells exhibited similar telomerase response to radiation and its regulation was assessed at transcriptional and post-translational levels in K562 cells. Next step was the evaluation of the upstream signaling pathways leading to changes in TA using kinetics and specific inhibitors. RESULTS: Radiation up-regulated TA in dose-dependent manner only in cancer cells. Telomerase was activated by phosphorylation by Akt and by cytoplasmic-nuclear shift. Transcriptional processes were not involved in TA. This telomerase regulation is mediated by Ras/phosphatidylinositol 3-kinase/Akt pathway. The canonical membrane effectors of irradiation (epidermal growth factor receptor, insulin-like growth factor-I receptor, and Ca2+ influx) were not involved in this process. CONCLUSIONS: Radiation up-regulates telomerase activity specifically in cancer cells. This study adds to accumulating evidence pointing to post-translational level as important mode of telomerase regulation. Telomerase activation due to radiation may be detrimental in treatment of cancer. Data described in this study may add to future interventions aiming at inhibition of telomerase activation during irradiation.

Correction: Premature ageing following allogeneic hematopoietic stem cell transplantation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Premature ageing following allogeneic hematopoietic stem cell transplantation.

Survivors of hematopoietic cell transplantation (HCT) have been shown to exhibit both clinical and biological features of accelerated ageing. Most studies used frailty measures, comorbidities for clinical assessment and several biological assessment of premature ageing. However, these tests are less suitable for age determination of individual patients. Recently, DNA methylation has emerged as a novel test to measure cellular age. In the present study, we assessed ageing in a cohort of 26 survivors of allogeneic HCT by frailty tests comprising the handgrip and 6 min walk tests and by biological tests including DNA methylation, telomere length and expression of p16INK4A and serum levels of IL-6. DNA methylation was evaluated both in blood and buccal epithelial cells. Physiological reserve was markedly reduced in transplant survivors, reflected by 6 min walk test. Increased IL-6 serum levels and p16ink4A correlated with accelerated ageing. Overall, the measured age of donor blood cells was significantly higher than these blood cells residing in their respective donors, as reflected by DNA methylation and by buccal epithelium methylation status. These clinical and biological observations suggest that allogeneic HCT is associated with accelerated ageing.

Peripheral blood telomere alterations in ground glass opacity (GGO) lesions may suggest malignancy.

A ground glass opacity (GGO) lung lesion may represent early stage adenocarcinoma, which has an excellent prognosis upon prompt surgical resection. However, GGO lesions have broad differential diagnoses, including both benign and malignant lesions. Our objective was to study telomere length and telomerase activity in patients with suspected lung cancer in which GGO was the predominant radiographic feature. Knowledge of telomere biology may help distinguish malignant from benign radiographic lesions and guide risk assessment of these lesions. Peripheral blood samples were taken from 22 patients with suspected adenocarcinoma with the GGO radiographic presentation. Multidisciplinary discussion confirmed the need for surgery in all cases. We used an age and gender-matched group without known lung disease as a control. Telomere length and aggregates were assessed by quantitative fluorescence in situ hybridization (QFISH) and quantitative PCR. Cell senescence was evaluated by senescence-associated heterochromatin foci. Subjects with GGO lesions had a higher percentage of lymphocytes with shorter telomeres (Q-FISH, P = 0.003). Furthermore, relative telomere length was also reduced among the GGO cases (qPCR, P < 0.05). Increased senescence was observed in the GGO group compared to controls (P < 0.001), with significant correlation between the senescence-associated heterochromatin foci and aggregate formation (r = -0.7 and r = -0.44 for cases and controls, respectively). In conclusion, patients with resectable early adenocarcinoma demonstrate abnormal telomere length and cell senescence in peripheral blood leukocytes compared to control subjects. Abnormal telomere biology in the peripheral blood may increase suspicion of early adenocarcinoma among patients with GGO lesions.

The Effect of Ethanol on Telomere Dynamics and Regulation in Human Cells.

Telomeres (TLs) protect chromosome ends from chromosomal fusion and degradation, thus conferring genomic stability, and play crucial roles in cellular aging and disease. Recent studies have found a correlation between environmental, physiological and even mental stresses on TL dynamics in humans. However, the causal relationship between stress and TL length and the molecular mechanisms underlying that relationship are far from being understood. This study describes the effect of moderate concentrations of ethanol, equivalent to social drinking, on human TL dynamics and partially elucidates the mechanism mediating this effect. The exposure of Immortalized human foreskin fibroblast, primary human foreskin fibroblast and human hepatocellular carcinoma cells to 25 mM ethanol for one week moderately shortened telomeres in all cells. Similar TL shortening was obtained following cells' exposure to 25 µM acetaldehyde (AcH) and to a much lower extent after exposure to 4-methylpyrazolean, an inhibitor of alcoholdehydrogenase, suggesting that AcH plays a key role in ethanol-dependent telomere shortening. Telomerase activity was not involved in this effect. TRF2 and several TRF2 binding proteins increased their binding to TLs after ethanol treatment, implying their involvement in this effect. The methylation status of several sub-telomeric regions increased in response to EtOH exposure. Gene expression profiling showed distinct patterns in cells treated with EtOH and in cells recovered from EtOH. In addition to cellular ageing, the described telomere shortening may contribute to the carcinogenic potential of acute alcohol consumption; both are associated with the shortening of TLs and provide new insights regarding the moderate consumption of alcohol referred to as "social drinking."

Tumor cells derived exosomes contain hTERT mRNA and transform nonmalignant fibroblasts into telomerase positive cells.

Exosomes are small (30-100nm) vesicles secreted from all cell types serving as inter-cell communicators and affecting biological processes in "recipient" cells upon their uptake. The current study demonstrates for the first time that hTERT mRNA, the transcript of the enzyme telomerase, is shuttled from cancer cells via exosomes into telomerase negative fibroblasts, where it is translated into a fully active enzyme and transforms these cells into telomerase positive, thus creating a novel type of cells; non malignant cells with telomerase activity. All tested telomerase positive cells, including cancer cells and non malignant cells with overexpressed telomerase secreted exosomal hTERT mRNA in accordance with the endogenous levels of their hTERT mRNA and telomerase activity. Similarly exosomes isolated from sera of patients with pancreatic and lung cancer contained hTERT mRNA as well. Telomerase activity induced phenotypic changes in the recipient fibroblasts including increased proliferation, extension of life span and postponement of senescence. In addition, telomerase activity protected the fibroblasts from DNA damage induced by phleomycin and from apoptosis, indicating that also telomerase "extracurricular" activities are manifested in the recipient cells. The shuttle of telomerase from cancer cells into fibroblasts and the induction of these changes may contribute to the alterations of cancer microenvironment and its role in cancer. The described process has an obvious therapeutic potential which will be explored in further studies.

BRCA1/2 mutations perturb telomere biology: characterization of structural and functional abnormalities in vitro and in vivo.

BRCA1 mutation is associated with carcinogenesis, especially of breast tissue. Telomere maintenance is crucial for malignant transformation. Being a part of the DNA repair machinery, BRCA1 may be implicated in telomere biology. We explored the role of BRCA1 in telomere maintenance in lymphocytes of BRCA1/2 mutation carriers and in in vitro system by knocking down its expression in non-malignant breast epithelial cells.The results in both systems were similar. BRCA1/2 mutation caused perturbation of telomere homeostasis, shortening of the single stranded telomere overhang and increased the intercellular telomere length variability as well as the number of telomere free chromosomal ends and telomeric circles. These changes resulted in an increased DNA damage status. Telomerase activity, inducibility and expression remained unchanged. BRCA1 mutation resulted also in changes in the binding of shelterin proteins to telomeres. DNMT-1 levels were markedly reduced both in the carriers and in in vitro system. The methylation pattern of the sub-telomeric regions in carriers suggested hypomethylation in chromosome 10. The expression of a distinct set of genes was also changed, some of which may relate to pre-disposition to malignancy.These results show that BRCA gene products have a role in telomere length homeostasis. It is plausible that these perturbations contribute to malignant transformation in BRCA mutants.

Anti-proliferative activity of haloperidol in B16 mouse and human SK-MEL-28 melanoma cell lines.

Sigma receptors are present in cancer cell lines. The aim of the present study is to evaluate the anti-tumor activity of a series of sigma 1, sigma 2 and sigma 1/2 ligands in B16 melanoma cell lines. Proliferation, apoptosis, intracellular ATP content, cell cycle and molecular regulators were analyzed. Cell growth was determined using the sulforhodamine B (SRB) colorimetric cytotoxicity assay. Apoptosis was assessed by flow cytometry and DNA fragmentation, using ELISA cell death assay. ATP content was measured spectrofluorometrically and cell cycle analysis was performed by flow cytometry. The cytoplasmic and nuclear expression of cell cycle regulatory molecules, cyclin D and CDK2 (cyclin dependent kinase 2) were determined by Western blot analysis and quantified by densitometry. The sigma ligands in single digit micromolar concentrations inhibited B16 and multidrug-resistant B16 COL/R cell growth, leading to cell death at higher concentrations. The potency order was: haloperidol, reduced-haloperidol, ifenprodil tartrate, opipramol and carbetapentane citrate. B16 COL/R cells were to some extent, less sensitive to sigma ligands. Further studies have shown that the growth inhibitory effect of sigma ligands could be attributed to G1 arrest of the cell cycle, mediated by a marked decrease in cytoplasmic and nuclear cyclin D and CDK2 protein expression, though haloperidol induced loss of cell viability due to apoptosis. Sigma ligands induced an early decrease in ATP content. These data stimulated us to examine the combined anti-proliferative activity of haloperidol and the tyrosine kinase inhibitor imatinib mesylate (STI 571), on SK-MEL-28 human melanoma cells. Preliminary experiments demonstrated a marked synergistic interaction between the two agents.

Combined effect of aloe-emodin and chemotherapeutic agents on the proliferation of an adherent variant cell line of Merkel cell carcinoma.

Merkel cell carcinoma (MCC) has only limited sensitivity to chemotherapeutic agents. The aim of the study was to determine if members of the anthraquinone family could be used as adjuncts to increase the growth inhibiting effect of anticancer agents in MCC. An adherent variant of MCC was derived from a previously established MCC cell line suspension. Cells were characterized by immunocytochemical methods using specific antibodies against epithelial (low molecular weight cytokeratins and cytokeratin 20) and neuroendocrine (neuron-specific enolase, neurofilament protein, chromogranin A and synaptophysin) antigens. Emodin and aloe-emodin, members of the anthraquinone family, inhibited proliferation of the adherent MCC cells, with a slight advantage of aloe-emodin over emodin. Aloin had no effect on cell proliferation. The chemotherapeutic agents, cis-platinol (abiplastin), doxorubicin (adriablastin), and 5-fluorouracil, and the tyrosine kinase inhibitor STI 571, all independently inhibited the proliferation of adherent MCC cells. The addition of aloe-emodin potentiated their inhibitory effect, especially when low concentrations of the anticancer compounds were used. The antiproliferative action of STI 571 may be associated with the presence of anti-c-kit antibodies. The combined use of anticancer agents, especially at low concentrations, and aloe-emodin may be considered a preferable means for treating MCC.

Variable cytotoxicity of amifostine in malignant and non-malignant cell lines.

Amifostine is the best known radioprotector and chemoprotector which has already been incorporated into general oncology practice. However, the data regarding its action at the cellular level remain unclear. The present study examined the effect of amifostine with and without ionizing radiation on the growth of malignant and non-malignant cell lines. Amifostine was found to have a remarkable cytotoxic effect on malignant epithelial cell lines but a modest cytotoxic effect on malignant melanoma and non-malignant cell lines. It demonstrated an additive effect with radiation therapy on the malignant cell line and a variable effect on the non-malignant cell line. Endothelial cells were not affected by amifostine, but the myoblast cells showed a synergistic effect of amifostine and radiation. These findings demonstrate that the cytotoxic as well as the radioprotective effect of amifostine are cell-specific. Thus, caution should be exercised in the use of amifostine as a radioprotector, and it should be tested for each model of disease.