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Obstructive sleep apnea syndrome (OSA) is associated with cardiovascular morbidity in adults and children. NFκB activity is enhanced in circulating monocytes of adults with OSA, that decreases following positive pressure therapy. OSA children’s serum activates NFκB in a cell line. We hypothesized that OSA children’s serum can activate NFκB in cardiomyocytes (CM) and effect their viability. In order to explore the role played by NFκB in OSA cardiovascular pathophysiology, rat, mouse and human immortalized CM were exposed to human serum drawn from OSA children and matched controls. Increased expression of NFκB classical subunits p65/p50 as well as major morphological changes occurred in cardiomyocytes following OSA’s serum exposure. OSA children’s serum induced NFκB activity as measured by p65 nuclear translocation in immortalized human CM and rat cardiomyocytes as well as dense immunostaining of the nucleus. Trypan blue and XTT assays showed that OSA sera induced CM apoptosis. We conclude that NFκB is systemically activated in cardiomyocytes, who also demonstrate decreased viability and contractility following exposure to OSA serum. It supports the hypothesis NFκB plays a role in the evolution of cardiovascular morbidity in OSA. It may support the search for new therapeutic interventions controlling NFκB activation in OSA.

Background: The authors investigated the NF-κB inhibitory role of three Benzisothiazolone (BIT) derivatives (1, 2 and 3) in Hodgkin’s Lymphoma cells (L428) which constitutively express activated NF-κB. All three compounds showed dose-dependent NF-κB inhibition (78.3, 70.7 and 34.6%) in the luciferase reporter gene assay and were found cytotoxic at IC50 values of 3.3μg/ml, 4.35μg/ml and 13.8μg/ml, respectively by the XTT assay. BIT 1and BIT 2 (but not BIT 3) suppressed both NF-κB subunits p50 and p65 in cytoplasmic and nuclear extracts in a concentration-dependent manner. Furthermore, BIT 1 showed a moderate synergistic effect with the standard chemotherapy drugs etoposide and doxorubicin, whereas BIT 2 and 3 showed a moderate additive effect to antagonistic effect. Cisplatin exhibited an antagonist effect on all the compounds tested under various concentrations, except in the case of 1.56μg/ml of BIT 3 with 0.156μg/ml of cisplatin. The compounds also inhibited the migration of adherent human lung adenocarcinoma cells (A549) in vitro. We conclude that especially BIT 1 and BIT 2 have in vitro anti-inflammatory and anti-cancer activities, which can be further investigated for future potential therapeutic use. Methods: Inspired by the electrophilic sulfur in Nuphar alkaloids, monomeric and dimeric benzisothiazolones were synthesized from dithiodibenzoic acid and their NF-κB inhibitory role was explored. NF-κB inhibition and cytotoxicity of the synthesized derivatives were studied using luciferase reporter gene assay and XTTassay. Immunocytochemistry studies were performed using L428 cells. Cell migration assay was conducted using the A549 cell line. L428 cells were used to conduct combination studies and the results were plotted using CompuSyn software. Results: Benzisothiazolone derivatives exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. Potent compounds showed suppression of both NF-κB subunits p50 and p65 in a concentrationdependent manner, both in cytoplasmic and nuclear extracts. Combination studies suggest that benzisothiazolone derivatives possess a synergistic effect with etoposide and doxorubicin. Furthermore, the compounds also inhibited the migration of A549 cells. Conclusion: Benzisothiazolones bearing one or two electrophilic sulfur atoms as part of the heterocyclic framework exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. In addition, these derivatives also exhibited a synergistic effect with etoposide and doxorubicin along with the ability to inhibit the migration of A549 cells. Our study suggests that BIT-based new chemical entities could lead to potential anticancer agents.

A number of natural products with medicinal properties increase DNA cleavage mediated by type II topoisomerases. In an effort to identify additional natural compounds that affect the activity of human type II topoisomerases, a blind screen of a library of 341 Mediterranean plant extracts was conducted. Extracts from Nuphcir lutea, the yellow water lily, were identified in this screen. N. lutea has been used in traditional medicine by a variety of indigenous populations. The active compound in N. lutea, 6,6’-dihydroxythiobinupharidine, was found to enhance DNA cleavage mediated by human topoisomerase IIα and IIβ ~8-fold and ~3-fold, respectively. Mechanistic studies with topoisomerase IIα indicate that 6,6’-dihydroxythiobinupharidine is a “covalent poison” that acts by adducting the enzyme outside of the DNA cleavage-ligation active site and requires the N-terminal domain of the protein for its activity. Results suggest that some of the medicinal properties of N. lutea may result from the interactions between 6,6’-dihydroxythiobinupharidine and the human type II enzymes.

Cardiovascular morbidity is the leading cause of death of obstructive sleep apnea (OSA) syndrome patients. Nocturnal airway obstruction is associated with intermittent hypoxia (IH). In our previous work with cell lines, incubation with sera from OSA patients induced changes in cell morphology, NF-κB activation and decreased viability. A decrease in beating rate, contraction amplitude and a reduction in intracellular calcium signaling was also observed in human cardiomyocytes differentiated from human embryonic stem cells (hESC-CMs). We expanded these observations using a new controlled IH in vitro system on beating hESC-CMs. The Oxy-Cycler system was programed to generate IH cycles. Following IH, we detected the activation of Hif-1α as an indicator of hypoxia and nuclear NF-κB p65 and p50 subunits, representing pro-inflammatory activity. We also detected the secretion of inflammatory cytokines, such as MIF, PAI-1, MCP-1 and CXCL1, and demonstrated a decrease in beating rate of hESC-CMs following IH. IH induces the co-activation of inflammatory features together with cardiomyocyte alterations which are consistent with myocardial damage in OSA. This study provides an innovative approach for in vitro studies of OSA cardiovascular morbidity and supports the search for new pharmacological agents and molecular targets to improve diagnosis and treatment of patients.

Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to their multiple applications against human ailments. Phyto-active Nuphar extracts and their purified and synthetic derivatives have attracted the attention of ethnobotanists and biochemists. Here, we report that 6,6′-dihydroxythiobinupharidine (DTBN), purified from extracts of Nuphar lutea (L.) Sm. leaves, is an effective inhibitor of the kinase activity of members of the protein kinase C (PKC) family using in vitro and in silico approaches. We demonstrate that members of the conventional subfamily of PKCs, PKCα and PKCγ, were more sensitive to DTBN inhibition as compared to novel or atypical PKCs. Molecular docking analysis demonstrated the interaction of DTBN, with the kinase domain of PKCs depicting the best affinity towards conventional PKCs, in accordance with our in vitro kinase activity data. The current study reveals novel targets for DTBN activity, functioning as an inhibitor for PKCs kinase activity. Thus, this and other data indicate that DTBN modulates key cellular signal transduction pathways relevant to disease biology, including cancer.

Objectives Nupharidine (6,6'-Dihydroxythiobinupharidine), purified from the aquatic plant Nuphar lutea leaves (Water lily) prompts antimicrobial activity of immune cells. The aim of the study was to test the effect of Nupharidine on neutrophil function against Aggregatibacter actinomycetemcomitans, JP2 clone (Aa-JP2). Methods Neutrophils derived from the human cell line HL60 and human peripheral blood derived from aggressive periodontitis and periodontally healthy subjects were incubated with Nupharidine or vehicle and inoculated with JP2. Bacterial survival was tested using viable counts on blood agar (CFU's). Neutrophils' necrosis/apoptosis, reactive oxygen species (ROS) production, phagocytosis and neutrophil extracellular traps (NET) production following infection were tested, as well as markers of neutrophil priming. Results Nupharidine had no direct bactericidal effect on JP2, but it enhanced Aa-JP2 clearance by neutrophils. Nupharidine enhanced neutrophil phagocytosis, ROS production and NET formation during JP2 infection. Furthermore, Nupharidine enhanced the expression of certain markers of neutrophils priming, specifically iCAM1, DECTIN-2 and intracellular IL-1β. Conclusion Nupharidine was shown to promote neutrophil effector bactericidal functions, boosting Aa-JP2 clearance. The results point to the potential of Nupharidine as an adjunctive agent in the treatment of Aa-JP2 periodontitis, but this should be tested initially using pre-clinical and clinical studies.

In this study, we found that the measles virus (MV) can infect human-induced pluripotent stem cells (hiPSCs). Wild-type MV strains generally use human signaling lymphocyte activation molecule (SLAM; CD150) as a cellular receptor, while vaccine strains such as the Edmonston strain can use both CD150 and CD46 as receptors. It is not yet known how early in the embryonal differentiation stages these receptors are expressed. We established two hiPSCs (BGU-iPSCs and EMF-iPSCs) which express CD46 and CD150. Both cell types can be infected by MV to form persistent, noncytopathic cell lines that release infectious MV particles. Following MV persistent infection, BGU-iPSCs and EMF-iPSCs remain pluripotent and can differentiate in vitro into the three germ layers. This includes cells expressing the neuronal differentiation markers: NF68 and miRNA-124. Since the MV does not integrate into the cell's genome, it can be utilized as a vehicle to systematically introduce genes into iPSC, to dissect and to define factors regulating lineage differentiation.

Obstructive sleep apnea syndrome (OSAS) patients suffer from cardiovascular morbidity, which is the leading cause of death in this disease. Based on our previous work with transformed cell lines and primary rat cardiomyocytes, we determined that upon incubation with sera from pediatric OSAS patients, the cell’s morphology changes, NF-κB pathway is activated, and their beating rate and viability decreases. These results suggest an important link between OSAS, systemic inflammatory signals and end-organ cardiovascular diseases. In this work, we confirmed and expanded these observations on a new in vitro system of beating human cardiomyocytes (CM) differentiated from human embryonic stem cells (hES). Our results show that incubation with pediatric OSAS sera, in contrast to sera from healthy children, induces over-expression of NF-κB p50 and p65 subunits, marked reduction in CMs beating rate, contraction amplitude and a strong reduction in intracellular calcium signal. The use of human CM cells derived from embryonic stem cells has not been previously reported in OSAS research. The results further support the hypothesis that NF-κB dependent inflammatory pathways play an important role in the evolution of cardiovascular morbidity in OSAS. This study uncovers a new model to investigate molecular and functional aspects of cardiovascular pathology in OSAS.

The specificity of inhibition by 6,6′-dihydroxythiobinupharidine (DTBN) on cysteine proteases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural-steric and biochemical differences. Cathepsin S (IC50 = 3.2 μM) was most sensitive to inhibition by DTBN compared to Cathepsin B, L and papain (IC50 = 1359.4, 13.2 and 70.4 μM respectively). DTBN is inactive for the inhibition of Mpro of SARS-CoV-2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close proximity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins-DTBN complexes, respectively. The lack of reactivity of DTBN against SARS-CoV-2 Mpro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of Mpro. Thus, gradations in reactivity among the tested Cathepsins may be conducive for a mechanism-based search for derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.

Ovarian cancer (OC) is the second most common type of gynecological malignancy, it has poor survival rates and is frequently (&gt, 75%) diagnosed at an advanced stage. Platinum-based chemotherapy, with, e.g., carboplatin, is the standard of care for OC, but toxicity and acquired resistance to therapy have proven challenging. Despite advances in OC diagnosis and treatment, approximately 85% of patients will experience relapse, mainly due to chemoresistance. The latter is attributed to alterations in the cancer cells and is also mediated by tumor microenvironment (TME). Recently, we reported the synthesis of a platinum (IV) prodrug that exhibits equal potency toward platinum-sensitive and resistant OC cell lines. Here, we investigated the effect of TME on platinum sensitivity. Co-culture of OC cells with murine or human mesenchymal stem cells (MS-5 and HS-5, respectively) rendered them resistant to chemotherapeutic agents, including platinum, paclitaxel and colchicine. Platinum resistance was also conferred by co-culture with differentiated murine adipocyte progenitor cells. Exposure of OC cells to chemotherapeutic agents resulted in activation of phospho-ERK1/2. Co-culture with MS-5, which conferred drug resistance, was accompanied by blockage of phospho-ERK1/2 activation. The flavonoids fisetin and quercetin were active in restoring ERK phosphorylation, as well as sensitivity to platinum compounds. Exposure of OC cells to cobimetinib&mdash, a MEK1 inhibitor that also inhibits extracellular signal-regulated kinase (ERK) phosphorylation&mdash, which resulted in reduced sensitivity to the platinum compound. This suggests that ERK activity is involved in mediating the function of flavonoids in restoring platinum sensitivity to OC co-cultured with cellular components of the TME. Our data show the potential of combining flavonoids with standard therapy to restore drug sensitivity to OC cells and overcome TME-mediated platinum drug resistance.

Many viruses can establish non-cytolytic, chronic infections in host cells. Beyond the intrinsically interesting questions of how this long-term parasitism is achieved, persistently infected cells can be useful to study virus-host interactions. MicroRNAs (miRNAs) are a class of noncoding RNAs transcribed from the genomes of all multicellular organisms and some viruses. Individual miRNAs may regulate several hundred genes. In this research we have studied the expression of a selective group of host-cell encoded miRNAs, as expressed in a Respiratory Syncytial Virus (RSV) persistently infected HEp-2 cell line (HEp-2 + RSV-GFP). The RSV is a virus that does not encode miRNAs in its genome. Our study shows that Dicer is down regulated, miRNA’s 146a-5p is strongly up-regulated and miRNAs 345-5p, let-7c-5p and miRNA’s-221 are down-regulated in HEp-2 + RSV-GFP cells. Correspondingly, changes in the miRNA 146a-5p and he sequences of the reference genes are miRNA 345-5p respective miRNAs target proteins: HSP-70 and p21, were observed. Thus, RSV persistent viral infection induces unique patterns of miRNA’s expression with relevance to how the virus regulates the host cell response to infection.

Measles virus (MV) infects a variety of lymphoid and non-lymphoid peripheral organs. However, in rare cases, the virus can persistently infect cells within the central nervous system. Although some of the factors that allow MV to persist are known, the contribution of host cell-encoded microRNAs (miRNA) have not been described. MiRNAs are a class of noncoding RNAs transcribed from genomes of all multicellular organisms and some viruses, which regulate gene expression in a sequence-specific manner. We have studied the contribution of host cell-encoded miRNAs to the establishment of MV persistent infection in human neuroblastoma cells. Persistent MV infection was accompanied by differences in the expression profile and levels of several host cell-encoded microRNAs as compared to uninfected cells. MV persistence infection of a human neuroblastoma cell line (UKF-NB-MV), exhibit high miRNA-124 expression, and reduced expression of cyclin dependent kinase 6 (CDK6), a known target of miRNA-124, resulting in slower cell division but not cell death. By contrast, acute MV infection of UKF-NB cells did not result in increased miRNA-124 levels or CDK6 reduction. Ectopic overexpression of miRNA-124 affected cell viability only in UKF-NB-MV cells, causing cell death; implying that miRNA-124 over expression can sensitize cells to death only in the presence of MV persistent infection. To determine if miRNA-124 directly contributes to the establishment of MV persistence, UKF-NB cells overexpressing miRNA-124 were acutely infected, resulting in establishment of persistently infected colonies. We propose that miRNA-124 triggers a CDK6-dependent decrease in cell proliferation, which facilitates the establishment of MV persistence in neuroblastoma cells. To our knowledge, this is the first report to describe the role of a specific miRNA in MV persistence.

In recent years it has become evident that bacteria can modulate signaling pathways in host cells through the secretion of small signaling molecules. We have evaluated the cytotoxic effects and NF-κB inhibitory activities of a panel of quorum sensing molecules and their reactive analogs on Hodgkin's lymphoma cells (L428). We found that several molecules inhibited NF-κB signaling in a dose dependent manner. Three inhibitors (ITC-12, ITC-Cl and Br-Furanone) showed 50% NF-κB inhibition at concentrations less than 10 µM (4.1 µM, 12.8 µM and 9.9 µM, respectively). Furthermore, all three molecules displayed cytotoxic effects against L428 cells with IC50 values of 12.4 µM, 18.3 µM and 3.1 µM respectively after 48 h incubation. They also showed inhibition of A549 adenocarcinoma cell migration at low concentrations 5.6 µM, 2.6 µM and 7.9 µM respectively. Further analysis showed that these molecules significantly decrease the degree of expression of proteins of NF-κB subunits p50, p65 and RelB both in cytosolic and nuclear fractions. This confirms that these compounds have the potential to modulate the NF-κB pathway by suppressing their subunits and thus exhibit cytotoxicity and inactivation of NF-κB signaling in Hodgkin's lymphoma cells.

O(6)-methylguanine-DNA methyltransferase (MGMT), is a DNA repair enzyme that recognizes O(6)-alkylated guanine, a base analog resulting from treatment with alkylating agents. O(6)-6-thioguanine (6-TG) is used clinically to treat malignant as well as inflammatory diseases. Although MGMT participates in resistance to alkylating agents, it has not been shown to be involved in resistance of tumors to 6-TG. In this study we used a human melanoma cell line (GA) and its selected 6-TG drug resistant variant (GA-6-TG) to investigate whether MGMT plays a role in determining the drug resistant phenotype of GA-6-TG cells. We showed that GA-6-TG resistant cells express about three fold more MGMT protein and mRNA than GA cells. Treatment with 6-TG diminishes significantly MGMT amounts in both cell lines. Increased amounts of MGMT in resistant cells, are consistent with hypermethylation of the MGMT gene coding-region. Pretreatment of cells with the MGMT inhibitor O6 benzyl guanine, resulted in sensitization of GA-6-TG cells to 6-TG. Taken together, our data suggests that MGMT is associated with 6-TG drug resistance. In analogy to patients treated with alkylating agents, patients with tumors containing increased MGMT amounts, may be more resistant to 6-TG and therefore may benefit from treatment with MGMT inhibitors.

In recent years the frequency of nonsmokers among lung cancer patients has increased to 10% to 15%. The measles virus has rarely been evoked as an etiological agent in malignant tumors and its role in carcinogenesis remains doubtful. It has been suggested that measles virus phosphoprotein may inhibit ubiquitination of Pirh2, which has been reported to be overexpressed in lung carcinoma and is responsible for degrading the cell cycle regulator p53. The authors conducted a clinicopathological study of newly diagnosed patients with non-small cell lung carcinoma of all stages seen in a 10-year period. Immunohistochemical studies for measles virus antigens, p53, and Pirh2 were performed using the avidin-biotin peroxidase complex. The authors found expression of measles virus antigens in 54 of 65 cases of non-small cell lung carcinoma. This finding is associated with the older age of the patients and with expression of Pirh2. The presence of Pirh2 itself was associated with improved survival.

The oncogenes c-myc and c-ras are known to elicit a cooperative tumorigenicity. In this study we investigated their role in the pathogenesis of Hodgkin's disease. The expression of these oncogenes was determined in Hodgkin's disease patients by avidin-biotin peroxidase complex immunohistochemical staining and was compared to their expression in patients with non-Hodgkin's lymphomas and inflammatory reactive lymph nodes. Of 29 examined patients with different histological types of Hodgkin's disease, 21 (72.4%) showed an elevated expression of c-myc and 28 (96.5%) of c-ras. Although this expression was marked especially in the neoplastic Reed-Sternberg cells, it was also noted in the numerous reactive cells present in the involved lymph nodes. By contrast, a much lower frequency of increased expression of these oncogenes was recorded in 19 patients with different grades of non-Hodgkin's lymphoma and in 29 patients with inflammatory reactive lymph nodes. The elevated expression of c-myc and c-ras in the neoplastic Reed-Sternberg cells may reflect an oncogenic event that directly activates these genes. However, their increased expression in the surrounding non-neoplastic cells probably results from signal transduction induced by certain growth-promoting factors possibly released by the Reed-Sternberg cells and that act paracrinally to stimulate the proliferation of the neighboring cells. Furthermore, the continuous c-ras elevation may impair the normal cell cycle control and thereby promote mutagenesis and overt malignancy.

We investigated the ability of FTIR-microscopy to define spectral changes between drug-sensitive and drug-resistant human melanoma cells. As a model system, a resistant melanoma cell line (GAC) was selected with cisplatin from parental (GA) cells. Using Fourier transform infrared spectroscopy (FTIR) we investigated the ability to differentiate between the resistant variant derived from the sensitive parental cell line, in the absence of cisplatin. We determined and validated spectral parameters (biomarkers) that differentiated between the two cell lines. By applying the principal component analysis (PCA) model, we reduced the original data size to six principal components. We detected a significant and consistent increase in the cell's DNA/RNA ratio as well as an increase in the lipid/protein ratio in the resistant cells. These results strongly support the potential of developing FTIR microspectroscopy as a simple, reagent-free method for the identification of drug-resistant cells. Rapid detection of tumors resistant to a particular drug, should contribute to the ability of the physician to choose an effective treatment protocol.

The Hodgkin-Reed-Sternberg (HRS) malignant cells in Hodgkin's lymphoma (HL) originate from germinal center B lymphocytes that did not undergo apoptosis. Protein Kinase C (PKC), a family of serine/threonine kinases, plays a crucial role in signal transduction modulating cell growth, differentiation and apoptosis. Here, we report the expression of PKC isoforms in two HL-derived cell lines, L428 and KMH2 and their correlation with drug resistance to CPT and doxorubicin. Among the PKC isoforms examined, only PKCeta and PKCbetaII were preferentially expressed in the drug resistant L428 cells. We have shown correlation between the response to apoptosis of L428 and KMH2 cells and PKCeta expression in these cell lines. In order to directly demonstrate a role for PKCeta in apoptosis, its expression was knocked-down by siRNA in the resistant L428 cells. Downregulation of PKCeta rendered L428 cells more sensitive to doxorubicin and CPT. Furthermore, PKCeta knocked-down cells showed increased PARP-1 cleavage, cytochrome c release and caspase 7 activation. It appears that PKCeta functions as an anti-apoptotic protein in HL-derived cell lines, and as we show here that it is also expressed in HRS of HL biopsies, it may have therapeutic relevance in HL. Thus, PKCeta could provide a new target aimed to reduce resistance to anti-cancer treatments of HL and other cancer patients.

In recent years several reports have been published describing dogs' ability to detect, by scent, patients with cancer. This ability is based on the sniffing of volatile organic elements that are secreted by malignant cells or react to them.To evaluate the ability of trained dogs to detect breast cancer cell cultures (MCF7) compared to the control pseudo-normal keratinocyte cell line (HaCaT), and to detect melanoma (BG) and type 2 epithelial lung carcinoma (A549) malignant cell cultures to which they were not previously exposed in the course of their training.Cell cultures were prepared in a standard manner. Two Belgian Shepherd dogs were trained and then tested in a single-blind test (for dogs and trainers) on their ability to detect the "target specimen," a MCF7 breast cancer cell culture. Following this, the ability of the dogs to detect cancer cell cultures that they were not previously exposed to (i.e., A549, BG) was tested. In each test round, four specimens placed in identical blocks were arranged in a line with one meter between them: one target specimen (MCF7, A549, BG), two control specimens (HaCaT), and a sample containing cell culture medium only.The two dogs picked out all the target specimens of MCF7 breast cancer cell cultures that they were trained to detect (10/10) as well as all the target specimens that they were not previously exposed to [A549 (5/5) and BG (5/5)], but did not pick out the control specimens or the cell culture medium. Thus, the sensitivity, specificity, and positive and negative predictive values for both dogs were 100%.The results of this study support the assumption that cancer cells have a unique odor pattern, and that this odor pattern is common to different types of cancer.

Hodgkin’s Lymphoma (HL) is one of the most prevailing malignancies in young adults. Reed–Sternberg (RS) cells in HL have distinctive large cell morphology, are characteristic of the disease and their presence is essential for diagnosis. Enlarged cells are one of the hallmarks of senescence, but whether RS cells are senescent has not been previously investigated. Here we show that RS cells have characteristics of senescent cells; RS cells in HL biopsies specifically express the senescence markers and cell cycle inhibitors p21Cip1 and p16INK4a and are negative for the proliferation marker Ki-67, suggesting that these cells have ceased to proliferate. Moreover, the RS-like cells in HL lines, stained specifically for senescence-associated β-galactosidase (SA-β-gal). Oxidative stress promoted senescence in these cells as demonstrated by their staining for p21Cip1, p16INK4a, p53 and γH2AX. Senescent cells produce copious amounts of inflammatory cytokines termed ‘senescence-associated secretory phenotype’ (SASP), primarily regulated by Nuclear Factor κB (NF-κB). Indeed, we show that NF-κB activity and NF-κB-dependent cytokines production (e.g., IL-6, TNF-α, GM-CSF) were elevated in RS-like cells. Furthermore, NF-κB inhibitors, JSH-23 and curcumin reduced IL-6 secretion from RS-like cells. Thus, defining RS cells as senescent offers new insights on the origin of the proinflammatory microenvironment in HL.

This adherence study was performed to clarify the trafficking of Hodgkin–Reed–Sternberg (HRS) cells in Hodgkin's disease (HD) and thus try to unravel the peculiar pathways of dissemination in the early stages of this malignant neoplasm. Using non-neoplastic human necropsy or biopsy lymph node as well as tonsillar tissue sections, we have studied the adherence of the KMH-2 and L-428 HD-derived cell lines and have compared it to that of peripheral blood lymphocytes (PBLs). In necropsy lymph nodes, cell lines predominantly adhered to sinuses and paracortex, whilst adhered PBLs were distributed more widely. In biopsy lymph nodes, adhesion to high endothelial venules (HEVs) was rarely observed, whilst cell lines were found to adhere to sinuses. Inhibition by EDTA pretreatment affected adherence to HEVs as well as to sinuses and paracortex to a similar degree. Our findings point to the possible importance of the lymph node sinuses and paracortex in relation to homing of the HRS cells and their dissemination during the early stages of HD. The results suggest a significant primary role of the extracellular matrix of the paracortex and sinuses in the homing of HRS cells, with the HEVs of only secondary importance.

The quest for an infectious agent that may account for cases of Hodgkin's disease (HD) especially in young adults has proven vain until lately. We have recently reported findings that suggested the presence of measles virus (MV) antigens and MV RNA in the tissues of patients with HD. Support for an association between MV and HD has been provided by recent epidemiological findings relating the occurrence of HD to exposure to measles in pregnancy and the perinatal period. We now present further evidence of this putative association based on immunohistochemical, reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridisation studies (ISH) on HD tissues. Biopsies from 82 (54.3%) of our cohort of 154 patients showed a positive immunostain with at least two of the anti-measles antibodies used. Latent membrane protein-1 immunostaining for Epstein-Barr virus was positive in 46 (31.1%) of the patients examined. Reverse transcriptase-PCR and ISH for measles RNA were positive in seven and 10 of 28 patients, respectively. Preliminary clinicopathological associations between MV and HD are noted in this study, but no causal relationship can be claimed at this stage.

Objective The CD15 (Lewis x) cell surface oligosaccharide moiety is expressed in a variety of normal and tumor cells and recognized by selectins. The detection of CD15 on malignant Hodgkin-Reed-Sternberg (HRS) cells serves as a diagnostic marker of Hodgkin's lymphoma (HL). Retrospective studies suggest that the expression of nonsialylated CD15 molecules on HRS cells has a positive prognostic value while presence of sialylated CD15 may correlate with a poor outcome. However, the relevance of the CD15 antigen expression to the pathobiology of the disease is not clear. In this work, we studied the contribution of CD15 to cell adhesion and the activation of signaling cascades in two HL-derived cell lines, KMH-2 and L428. Methods Immobilized anti-CD15 monoclonal antibodies and recombinant E- and P-selectins were used to activate KMH-2 and L428 cells. Immunoblotting, immunoprecipitation, and the electrophoretic mobility shift assay were performed to detect tyrosine phosphorylation of c-Cbl, c-Jun nuclear translocation, and AP-1 DNA binding. Results Treatment of cells with antibodies against the sialylated and nonsialylated forms of CD15, or with immobilized selectins, induced changes in cell morphology. Tyrosine phosphorylation of c-Cbl, together with tyrosine phosphorylation of multiple protein substrates, was also induced. In addition, binding of the CD15 molecules induced nuclear translocation of c-Jun and an increase in AP-1 DNA binding activity. Conclusions We suggest that CD15 has a dual physiological role, both as an adhesion molecule recognized by selectins and as a regulatory molecule upstream to specific intracellular signaling cascades with implications to the pathogenesis of HL.

Epidemiologic and molecular investigations of Hodgkin's disease (HD) suggest a strong infectious association. The Epstein-Barr virus (EBV), together with its viral proteins, is expressed in Hodgkin-Reed-Sternberg (HRS) cells in the lymph nodes involved by HD. EBV is more likely to be related to childhood and older adult cases of HD and is much less frequently expressed in young adult HD patients, the group most expected to be associated with an infectious agent. In addition, the "hit and run" theory of EBV infection remains speculative and no other lymphotropic viruses studied to date seem to satisfy the quest for a new candidate virus in young adults with HD. We have recently found preliminary evidence suggesting a possible association between the measles virus (MV) and HD. This evidence is the subject of the present review.

We present a retrospective clinicopathological study on the significance of the histologic type of classical Hodgkin's disease (HD) in a cohort of patients from southern Israel. This was performed to critically evaluate the generally accepted view that classical HD is a single clinicopathological entity and the resultant impression that its segregation into four different histologic types remains essential only for the pathologist in his diagnostic endeavor. We confirmed the existence of a uniform response of nodular sclerosis (NS), and mixed cellularity-lymphocyte depletion (MC-LD)-HD to treatment, consideration being given to other classical prognostic factors. We also accept the fact that histological type is not a significant independent factor in terms of survival. Our findings, however, do suggest that NS-HD, on the one hand, and MC-LD-HD, on the other, are distinct biologic entities. Cases of NS differ significantly from those of MC-LD-HD with regard to sex and age distribution, and in the expression of several antigens and gene products, including sialylated-CD15, CD30, LMP1 and the p53 and mdm-2 gene products.

Oxidative stress plays a pivotal role in the development of human diseases. Reactive oxygen species (ROS) that includes hydrogen peroxide, hyphochlorus acid, superoxide anion, singlet oxygen, lipid peroxides, hypochlorite and hydroxyl radical are involved in growth, differentiation, progression and death of the cell. They can react with membrane lipids, nucleic acids, proteins, enzymes and other small molecules. Low concentrations of ROS has an indispensable role in intracellular signalling and defence against pathogens, while, higher amounts of ROS play a role in number of human diseases, including arthritis, cancer, diabetes, atherosclerosis, ischemia, failures in immunity and endocrine functions. Antioxidants presumably act as safeguard against the accumulation of ROS and their elimination from the system. The aim of this review is to highlight advances in understanding of the ROS and also to summarize the detailed impact and involvement of antioxidants in selected human diseases.

Study objectives Childhood obstructive sleep apnea syndrome (OSAS) is associated with an elevation of inflammatory markers such as C-reactive protein (CRP) that correlates with specific morbidities and subsides following intervention. In adults, OSAS is associated with activation of the transcription factor nuclear factor kappa B (NF-kB). We explored the mechanisms underlying NF-kB activation, based on the hypothesis that specific NF-kB signaling is activated in children with OSAS. Design Adenoid and tonsillar tissues from children with OSAS and matched controls were immunostained against NF-kB classical (p65 and p50) and alternative (RelB and p52) pathway subunits, and NF-kB-dependent cytokines: interleukin (IL)- 1α, IL-1β, tumor necrosis factor-α, and IL-8). Serum CRP levels were measured in all subjects. NF-kB induction was evaluated by a luciferase-NF-kB reporter assay in L428 cells constitutively expressing NF-kB and in Jurkat cells with inducible NF-kB expression. p65 translocation to the nucleus, reflecting NF-kB activation, was measured in cells expressing fluorescent NF-kB-p65-GFP (green fluorescent protein). Setting Sleep research laboratory. Patients or participants Twenty-five children with OSAS and 24 without OSAS. Interventions N/A. Measurements and results Higher expression of IL-1α and classical NF-kB subunits p65 and p50 was observed in adenoids and tonsils of children with OSAS. Patient serum induced NF-kB activity, as measured by a luciferase-NF-kB reporter assay and by induction of p65 nuclear translocation in cells permanently transfected with GFP-p65 plasmid. IL-1β showed increased epithelial expression in OSAS tissues. Conclusions Nuclear factor kappa B is locally and systemically activated in children with obstructive sleep apnea syndrome. This observation may motivate the search for new anti-inflammatory strategies for controlling nuclear factor kappa B activation in obstructive sleep apnea syndrome.

In this study, the composition and the role of membrane glycoproteins in phagocytosis were determined in G6PD deficient RBCs. G6PD deficient RBCs were recognized and significantly phagocytosed by murine macrophages, without pre-exposure to oxidants in vivo. Phagocytosis was partially (60%) inhibited by incubating macrophages with either galactose or mannose, or by incubating RBCs with beta-galactosidase, indicating the involvement of lectin-like receptors in the recognition of G6PD deficient RBCs. Membrane glycoproteins on G6PD deficient cells were detected by binding of Con A to both intact RBCs and to purified membrane proteins. The results demonstrated modifications in the glycoprotein pattern of G6PD deficient RBCs compared to untreated controls. These included reduction in the amounts of several high molecular weight glycoproteins and appearance of lower molecular weight bands. These results suggest that G6PD deficient RBCs undergo glycoprotein modifications, which may lead to premature removal from circulation, even in non-acute hemolysis.

Immunosuppressive acidic protein (IAP) has been described as a tumor marker in a number of malignant diseases. To evaluate the clinical importance of IAP in breast cancer patients, IAP serum level was determined in 75 breast cancer patients, using single radial immunodiffusion. Serum samples were also tested for CA 15-3. Cut off value for IAP was determined according to IAP serum level in 50 patients with benign, non inflammatory, diseases, and was set as 725 microgram/ml. Mean IAP serum level (623 mcg/ml) and positivity rate (20%) in 24 breast cancer patients with active disease were similar to those in 51 breast cancer patients with no evidence of disease (590 mcg/ml and 18%). The mean CA 15-3 serum level and positivity rate were significantly higher in patients with active disease (200 units/ml, 67%), compared to patients with no evidence of disease (18.3 units/ml, 6%). In our experience IAP was not found to be an effective tumor marker in breast cancer.

The nuclear factor κB (NF-κB) family of transcription factors participates in the regulation of genes involved in innate- and adaptive-immune responses, cell death and inflammation. The involvement of the Protein kinase C (PKC) family in the regulation of NF-κB in inflammation and immune-related signaling has been extensively studied. However, not much is known on the role of PKC in NF-κB regulation in response to DNA damage. Here we demonstrate for the first time that PKC-eta (PKCη) regulates NF-κB upstream signaling by activating the IκB kinase (IKK) and the degradation of IκB. Furthermore, PKCη enhances the nuclear translocation and transactivation of NF-κB under non-stressed conditions and in response to the anticancer drug camptothecin. We and others have previously shown that PKCη confers protection against DNA damage-induced apoptosis. Our present study suggests that PKCη is involved in NF-κB signaling leading to drug resistance.

Breast cancer (BC) is the most common cancer in women in Western countries, showing a bimodal age distribution with peaks at 50 and 70 years. Multiple factors are encountered in the etiology of BC, including hereditary and hormonal causes. A few viruses, including MMTV, EBV and HPV have been reported to be associated with BC. This group previously described the presence of the measles virus (MV) in biopsies of patients with Hodgkin's lymphoma. The present study explored MV antigens in BC patients.A total of 131 patients with invasive BC diagnosed during the years 1998-2005 were studied using immunohistochemistry (IHC) for the presence of MV antigens, hemagglutinin and nucleoprotein. Clinicopathological parameters included age, stage, histological grade and the expression of estrogen and progesterone receptors, HER2/neu, p53, and Ki67.Hormone receptors and HER2/neu were positive in 54% and 18% of the tumors, respectively. Both MV antigens were detected in 64% of the tumors. All biopsies containing a DCIS component showed MV in DCIS in addition to invasive BC. In univariate analysis, MV correlated with estrogen receptor (p=0.018), low Ki67 index (40%, p=0.029), low or intermediate grade (p=0.037), age under 50 years (p=0.039), progesterone receptor (p=0.043) and overexpression of p53 (p=0.049). In multivariate analysis, only grade (p=0.011), p53 (p=0.03) and age (p=0.041) remained associated with MV.This study provides evidence for the presence of MV antigens in a relatively large proportion of BC patients. MV was associated with younger age, lower histological grade and overexpression of p53, suggesting that it may play a role in the development of BC.

Attenuated total reflection (ATR) spectroscopy is used as an in vitro optical approach for the diagnosis and characterization of cell and tissue pathology. In comparison with the more conventional FTIR microspectroscopy that relies on transmission of IR radiation, ATR spectroscopy uses the evanescent wave technique, which is a step forward toward in vivo research. The aim of the present investigation was to examine the potential of ATR spectroscopy to differentiate between drug-resistant and drug-sensitive melanoma cell lines. We studied two human melanoma parental cell lines, GA and BG, and their cisplatin-resistant counterparts, GAC and BGC, respectively, which were derived by survival selection with this anticancer drug. Cisplatin cytotoxicity was measured on the four cell lines, and their relative resistance to cisplatin was established: BGC > BG > GAC > GA. Different resistance mechanisms were noticed between the two parental groups in accordance with their spectrum. ATR spectra-based cluster analysis of the selective biomarkers, such as phosphate and RNA/DNA, were found useful in differentiating sensitive from resistant cells. Normalized and absolute values of the differences between spectra were employed to compare between the two parental groups. It was possible to predict the relative cisplatin resistance between the cell lines using the discriminant classifying function. The success rates in predicting cisplatin resistance in these cells was 88 and 81% for GA versus GAC and BG versus BGC, respectively. These results support the further development of the ATR technique as a simple, in vitro, reagent-free method to identify drug resistance in cancer cells.

We scrutinized the role of apoptosis of the Hodgkin-Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) and critically reviewed its features in the light of conflicting evidence. In this study, we found that tumor cells in this neoplasm showed inhibition of apoptosis in 55% of the 217 cHL cases only. It is also suggested that the two factors considered responsible for apoptosis inhibition in HRS cells, nuclear factor-kappaB and the latent membrane protein-1 of the Epstein-Barr virus, do not correlate with apoptosis inhibition, in contrast with the findings in the consensual pathogenetic scheme. The most significant association of HRS cell apoptosis was with p53, the negative expression of which related with a high apoptotic index (p = 0.001). These findings support our contention that the role of apoptosis in the HRS cells of Hodgkin lymphoma has not been completely elucidated and is at variance with that in the consensus.

Hodgkin's lymphoma is a distinct primary solid tumor of the immune system that shows wide variation in incidence among different geographic regions and among various races. It was previously suggested that susceptible people living in certain parts of Israel had a higher risk of HL because of exposure to unidentified environmental factors in these regions. Compared with other parts of Israel, these regions were characterized by a higher proportion of Israeli-born Jews.To study time trends in the incidence rate of HL in Israeli-born Jews of all age groups during the years 1960-2005.A total of 4812 Jewish cases of HL were reported to the Israel Cancer Registry during the study period 1960-2005. There has been a persistent increase in the age-standardized incidence rate of HL, all subtypes pooled, in Israeli-born Jews in both men and women. The age distribution pattern in both genders was bimodal in all periods. The highest incidence was observed in the 20-24 year age group: for women (9.13 per 100,000 per year) during the period 1988-1996, and for men (6.60 per 100,000 per year) during the period 1997-2005.The reported incidence level of HL in Israeli-born young adult Jews in Israel has increased in recent years to high levels compared with other western countries. Our findings suggest a cohort effect to unidentified factors affecting Israeli-born young adult Jews in Israel.

We screened thirty-four methanolic plant extracts for inhibition of the constitutive nuclear factor kappaB (NFkappaB) activity by a NFkappaB-luciferase reporter gene assay. Strong inhibition of NFkappaB activity was found in extracts of leaf and rhizome from Nuphar lutea L. SM. (Nuphar). The inhibitory action was narrowed down to a mixture of thionupharidines and/or thionuphlutidines that were identified in chromatography fractions by one- and two-dimensional NMR analysis. Dimeric sesquiterpene thioalkaloids were identified as the major components of the mixture. The Nuphar alkaloids mixture (NUP) showed a dose dependent inhibition of NFkappaB activity in a luciferase reporter gene assay as well as reduction of nuclear NFkappaB subunits expression as tested by western blots and immunohistochemistry. Decreased DNA binding was demonstrated in electro mobility shift assays. NUP inhibited both inducible and constitutive NFkappaB activation and affected the canonical and alternative pathways. Suppression of NFkappaB was not cell type specific. Induction of apoptosis by the alkaloid mixture was demonstrated by time-dependent and dose-dependent cleavage of procaspase-9 and PARP. Synergistic cytotoxicity of the active mixture with cisplatin and etoposide was demonstrated. Overall, our results show that NUP inhibits the NFkappaB pathway and acts as a sensitizer to conventional chemotherapy, enabling the search for its specific target and application against cancer and inflammation.

Leishmaniasis is a vector-borne disease caused by flagellated protozoan parasites of the genus Leishmania, which affects both humans and other mammals. Most of the available drugs against the disease are toxic and parasite resistance to some of the drugs has already developed. In the present study, the leishmanicidal activities of methanolic extracts of some Israeli plants have been evaluated in vitro, against the free-living promastigotes and intracellular amastigotes of Leishmania major. Of the 41 extracts examined, those of two plants (Nuphar luteaWithania somnifera) were highly effective (with a maximum inhibitory effect of50%), those of three other species (Pteris vittataSmyrnium olusatrumTrifolium clypeatum) were moderately effective (25%-50%) and another four extracts (Erodium malacoidesHyparrhenia hirtaThymelaea hirsutaPulicaria crispa) showed a marginal effect (15%-22%) against the parasites. Extracts of nine plant species therefore showed antileishmanial activity but only the extract of N. lutea, used at 1.25 microg/ml, eliminated all the intracellular parasites within 3 days of treatment, with no detectable toxicity to the host macrophages. The mean (S.D.) values recorded for the median inhibitory concentrations of this extract (IC50) against the promastigotes [2.0 (0.12) microg/ml] and amastigotes [0.65 (0.023) microg/ml] and the median lethal concentration (LD50) against macrophages [2.1 (0.096) microg/ml] were encouraging, giving a therapeutic selectivity index [LD50/IC50 for amastigotes)] of 3.23. The extract of N. lutea was, in fact, generally as effective as the paromomycin that was used as the 'gold standard' drug. These results indicate that N. lutea and probably also Withania somnifera might be potential sources of clinically useful, antileishmanial compounds.

Objectives To look for an association between the measles virus and endometrial carcinoma, the most frequent cancer of the female genital tract in our area. Study design Thirty-six of 49 patients with endometrial carcinoma were studied to detect fingerprints of the measles virus. Immunohistochemistry with the avidin–biotin complex method and in situ hybridization were used to demonstrate the association. The clinicopathological correlations were carried out to support a relationship between the virus and the cancer if any was found. Results Twenty-six of the 36 cases (72%) of endometrial cancer showed the presence of measles virus antigens in the tumor cells. Sixteen of 21 cases were positive for measles virus RNA by in situ hybridization. Although type I endometrial carcinoma was more positive for viral particles than type II, type II cancer, when allied with the measles virus, was more often associated with the depth of myometrial invasion and with death from tumor. Conclusions We demonstrate for the first time a link between endometrial cancer and the presence of viral antigens and RNA of the measles virus, although these findings do not necessarily signify a causal relationship between the cancer and the virus.

In this study, the role of Igs in the recognition and removal of oxidatively damaged human red blood cells (RBCs) was investigated. Phagocytosis of normal RBCs exposed to the oxidative hemolytic agent phenylhydrazine (Phz) and of glucose-6-phosphate dehydrogenase (G6PD)- deficient RBCs by murine macrophages was examined. A 40-fold increase in phagocytosis of RBCs treated with 3 mmol/L Phz was obtained both in the absence and presence of autologous serum, indicating that binding of autologous antibodies to the oxidized cells is not essential for phagocytosis. Yet, a basal number of IgG molecules was found to be present on the RBCs, as determined both by binding of 125I protein A and fluorescein isothiocyanate-antihuman Ig to the cells. Macrophage Fc receptors were found to be involved in the recognition of the RBCs, because phagocytosis was partially inhibited by incubating macrophages with bovine serum albumin (BSA) anti-BSA complexes, aIg (aggregated Igs), and anti-Fc receptor II monoclonal antibodies. Galactose/mannose inhibited phagocytosis of oxidized RBCs additively to aIg. Because phagocytosis was decreased when Phz-RBCs were incubated with F(ab')2 fragments of antihuman antibodies, it is suggested that the basal amount of Igs bound to the cells plays a role in the recognition of Phz- RBCs. G6PD-deficient RBCs were recognized and phagocytosed by murine macrophages without preexposure to oxidants in vitro (mean of 19 RBCs/100 macrophages). This phagocytosis was not affected by the addition of serum and was inhibited by incubating macrophages with galactose/mannose and the various Fc receptor blockers. A positive correlation between hemoglobin content and the number of cell-bound Igs to each patient erythrocytes was found. These results support the involvement of both an Fc and a lectin-like macrophage receptor in the recognition and phagocytosis of Phz-oxidized and G6PD-deficient RBCs and suggest opsonization as a possible physiologic process for the removal of severe damaged RBCs.

Changes in the definition of lymphocyte-depleted classic Hodgkin lymphoma (LDcHL) have recently led to reclassification of many cases as other pleomorphic lymphomas. We have set out to identify LD cases in our cohort of cHL patients and determine their clinical and biological characteristics properties. We defined the morphologic picture of LDcHL according to selected criteria and determined how its features differ from those of other subtypes of cHL. Twelve of 201 cHL patients (5.9%) were diagnosed as showing LDcHL histology, a higher percentage of LDcHL than in most recent series. The LD cases were most often positive for Epstein-Barr virus and for sialyl-CD15. Defining the cases as either reticular (eight) or as diffuse fibrosis (three) variants was critical to the diagnostic approach. We conclude that LDcHL may be a neglected entity.

Phenylhydrazine (Phz) is a powerful hemolytic agent which has several effects on both normal and G6PD deficient red blood cells (RBCs). We have studied the mechanism of removal of Phz-damaged human RBCs by murine macrophages. Phagocytosis of Phz-treated RBCs was found to be 50 RBCs/100 mac as compared to 2 RBCs/100 mac of the controls. EGTA and sodium azide inhibited the phagocytosis, indicating a requirement for both calcium ions and energy. Incubation of macrophages with sugars such as D-galactose or D-mannose reduced phagocytosis of Phz-treated RBCs by up to 60%, indicating the involvement of a macrophage lectin-like receptor in the recognition of Phz-treated RBCs. The presence of serum in the phagocytosis assay did not affect either phagocytosis of Phz-treated RBCs or inhibition by sugars. beta-Galactosidase, but not neuraminidase, treatment of RBCs caused a significant inhibition in phagocytosis of Phz-treated RBCs. These results suggest that galactosyl residues are exposed on RBC membrane during oxidation, probably not as a result of desialization. We conclude that Phz-treated RBCs are detected as damaged cells mainly due to sugar changes on their membrane, which are directly recognized by lectin-like receptors on the macrophages.

In the search for viruses in Hodgkin lymphoma (HL), it was reported that the measles virus (MV) can be detected in the Hodgkin-Reed/Sternberg (HRS) cells of a large fraction of cases from Israel by immunohistochemistry or in situ hybridisation, suggesting a potential role of this virus in HL. To extend and validate this report, we studied HL-derived cell lines and HRS cells microdissected from German and Israeli HL cases for the presence of MV RNA genome and transcripts, analysing three MV genes (nucleoprotein, matrix, haemaglutinin). A cell line infected with MV was used as a positive control for MV RNA detection. MV RNA was detectable down to 1 infected cell in a nested RT-PCR. Pools of microdissected HRS cells from 18 German and 7 Israeli classical HL (the latter reported to be positive for MV proteins in the previous study) were analysed for MV genome and transcripts. None of the viral genes was obtained in independent replicate experiments in any of the 25 HL cases. A Southern blot hybridisation performed with the second round PCR products further confirmed the negative results. Whole HL tissue sections were analysed to exclude MV in non-HRS cells, also yielding negative results. We also analysed four HL cell lines and showed that these are MV-negative, too. In this cohort of German and Israeli HL patients—including cases previously typed as MV-positive—and HL-derived cell lines, there was no evidence of MV genome in the HRS cells. © 2007 Wiley-Liss, Inc.

Malignant melanoma resistance to chemotherapy remains a major limitation to treatment. Our aim was to identify genes associated with drug resistance, in order to better understand the molecular events underlying the drug-resistant phenotype.A human melanoma cell line and its drug-resistant variants obtained by selection with MNNG or 6-thioguanine were used. Alterations in gene expression were characterized by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Prominent mRNA fragments present in selected variants and not in the parental cells were identified and characterized by cloning and sequencing. Differential expression was confirmed by real-time RT-PCR.Three functionally distinct transcriptional products were demonstrated: the chaperonin subunit TCP 1-zeta-6A (CCT6A), the hyaluronan receptor CD44 and LPPR-2, the lipid phosphate phosphatase-related protein type-2.Genes with altered expression were identified in drug-resistant variants. The identified molecules may provide new insights into the molecular basis for melanoma resistance to chemotherapy.

Fourier transform infrared microspectroscopy (FTIR-MSP) has shown promise as a technique for detection of abnormal cell proliferation and premalignant conditions. In the present study, we investigate the absorbance in the sensitive wavenumber region between 2800 and 3000 cm(-1), which has been known to be due to the antisymmetric and symmetric stretching vibrations of CH2 and CH3 groups of proteins and lipids. We report common biomarkers from this region that distinguish between normal and malignant tissues and cell lines. Based on our findings, we propose that the wavenumber region around 2800 to 3000 cm(-1) in the FTIR spectra of cells and tissues could provide valuable scientific evidence at the onset of premalignancy and may be used for ex vivo and in vitro detection of carcinogenesis. To further examine the utility of these markers in cancer diagnosis and management, they are tested successfully in monitoring the changes occurring in leukemia patients during chemotherapy.

Destabilization of the genome seems to be an important step in the generation of drug resistance. Since malignant melanoma is extremely resistant to chemotherapy, we used human melanoma cell lines as a model to investigate the putative role of genomic instability in the appearance of drug resistance. Drug-resistant variants were obtained with MNNG, BiCNU, doxorubicin and 6-thioguanine selection of melanoma cell lines. Genomic alterations in variant cells were detected by arbitrarily primed PCR of Alu-I digested DNA (Alu-I-AP-PCR). Two differential DNA bands from 6-TG-resistant cell variants were sequenced. One is homologous to intron 25 of the neural cell adhesion molecule L1 and the second to endogenous retroviral LTR sequences. We have shown that drug-resistant melanoma cell lines accumulate genomic alterations that are efficiently detected by Alu I-AP-PCR and that drug-resistant variants show genomic instability, including variations in LTR sequences, which may be associated with the appearance of the drug resistance phenotype.

Hodgkin's disease (HD) is an unusual malignant neoplasm, mainly because of the rarity of tumor cells in the diseased tissues, but also due to a relatively favorable response to treatment. In a previous study, we have shown a variable degree of apoptosis in lymph nodes from HD patients. We now looked for clinicopathological correlations of apoptosis with special emphasis on the prognosis in this disease. A retrospective study of 92 patients was carried out, using in situ end labelling of DNA fragments and an apoptosis detection kit. An apoptotic index (Al) was calculated in each case, as the percentage of apoptotic Hodgkin-Reed-Sternberg cells out of the total number of tumor cells in 10 selected high power fields. An association between a high Al and advanced stages was noted. A Kaplan-Meier analysis showed a negative correlation between Al and survival (p=0.05). In a multivariable analysis adjusting for Ann Arbor stage, a high Al carried a 3.27 fold risk of dying of HD (OR=3.27; Cl=0.89-11.94). However, in our limited cohort of HD patients, Al was not an independent prognostic factor. The results of this study confirm the important role played by apoptosis in HD and suggest that the apoptotic index is probably a negative prognostic marker in this disease. Its assessment in patients with HD may provide a new, important clinical tool.

I. Levin, B. Klein, S. Segal, A. Eyal, J. Gopas, J. Kaneti, M. Saadon, O. Kuperman. Expression of HLA class I-encoded cell surface antigens in transitional cell carcinoma of the urinary bladder.

The study examined the morphology and frequency of cell death occurring spontaneously in lymph nodes from patients with Hodgkin's disease. In addition to necrosis, which was infrequent and usually in patches, we document two cell types showing features of individual cell death: mummy cells end apoptotic cells. Mummy cells present no evidence of DNA fragmentation, but show electron microscopic features of "dark cells." Apoptotic Hodgkin-Reed-Sternberg cells are found frequently and are easier to demonstrate by in situ and labeling of fragmented DNA than by light microscopy only. In many cases phagocytosis of apoptotic cells is also documented. The significance of these findings to the limited number of Hodgkin-Reed-Sternberg cells in most cases of Hodgkin's disease is discussed.