Therapeutic resistance resulting from mutations in Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways.

Chemotherapy remains a commonly used therapeutic approach for many cancers. Indeed chemotherapy is relatively effective for treatment of certain cancers and it may be the only therapy (besides radiotherapy) that is appropriate for certain cancers. However, a common problem with chemotherapy is the development of drug resistance. Many studies on the mechanisms of drug resistance concentrated on the expression of membrane transporters and how they could be aberrantly regulated in drug resistant cells. Attempts were made to isolate specific inhibitors which could be used to treat drug resistant patients. Unfortunately most of these drug transporter inhibitors have not proven effective for therapy. Recently the possibilities of more specific, targeted therapies have sparked the interest of clinical and basic researchers as approaches to kill cancer cells. However, there are also problems associated with these targeted therapies. Two key signaling pathways involved in the regulation of cell growth are the Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways. Dysregulated signaling through these pathways is often the result of genetic alterations in critical components in these pathways as well as mutations in upstream growth factor receptors. Furthermore, these pathways may be activated by chemotherapeutic drugs and ionizing radiation. This review documents how their abnormal expression can contribute to drug resistance as well as resistance to targeted therapy. This review will discuss in detail PTEN regulation as this is a critical tumor suppressor gene frequently dysregulated in human cancer which contributes to therapy resistance. Controlling the expression of these pathways could improve cancer therapy and ameliorate human health.

Role of hypoxia and autophagy in MDA-MB-231 invasiveness.

Survival strategies adopted by tumor cells in response to a hypoxic stress include activation of hypoxia-inducible factor 1 (HIF-1) and autophagy. However, the importance and the function of each molecular response is not well defined. In the present study, we investigated invasiveness, migration, matrix metalloproteinases (MMPs) activity, and cell survival of MDA-MB-231 cells under normoxia, hypoxia, and hypoxia/reoxygenation (H/R). Moreover, to assess the importance of hypoxia and autophagy on the parameters studied, cells were either left untreated or treated with Chetomin (a selective inhibitor of HIF-1alpha) or trifluoperazine (TFP, an activator of autophagy). We found that hypoxia and H/R stimulated invasiveness and migration of MDA-MB-231 cells with an increased MMP-2 activity. Chetomin and TFP differently regulated the cellular behavior under the oxygenation conditions studied. In fact, Chetomin was most effective in inhibiting cell invasion, MMPs activity, and cell survival under hypoxia but not normoxia or H/R. By contrast, TFP inhibition of cell invasion, migration, and cell survival was independent from oxygenation conditions. TFP-induced autophagy was inhibited by light chain protein 3 (LC3) silencing or 3-methyladenine (3MA) treatment. In fact, LC3-silenced cells were able to invade in the presence of TFP without any GATE16 processing and p62 degradation. Immunofluorescence assay showed that LC3 silencing inhibited TFP-induced autophagosome formation. However, we also showed that both TPF treatment and LC3 silencing caused cytoskeleton impairments suggesting a possible interaction between LC3 and cytoskeleton components. In conclusion, our study shows that hypoxia and autophagy by acting on common (HIF-1alpha) or separate (MMPs, cytoskeleton) targets differently regulate cell invasion, MMPs activity, and survival.

Emerging MEK inhibitors.

IMPORTANCE OF THE FIELD: The Ras/Raf/MEK/ERK pathway is often activated by genetic alterations in upstream signaling molecules. Integral components of this pathway such as Ras and B-Raf are also activated by mutation. The Ras/Raf/MEK/ERK pathway has profound effects on proliferative, apoptotic and differentiation pathways. This pathway can often be effectively silenced by MEK inhibitors. AREAS COVERED BY THIS REVIEW: This review will discuss targeting of MEK which could lead to novel methods to control abnormal proliferation which arises in cancer and other proliferative diseases. This review will cover the scientific literature from 1980 to present and is a follow on from a review which focused on Emerging Raf Inhibitors published in this same review series. WHAT THE READER WILL GAIN: By reading this review the reader will understand the important roles that genetics play in the response of patients to MEK inhibitors, the potential of combining MEK inhibitors with other types of therapy, the prevention of cellular aging and the development of cancer stem cells. TAKE HOME MESSAGE: Targeting MEK has been shown to be effective in suppressing many important pathways involved in cell growth and the prevention of apoptosis. MEK inhibitors have many potential therapeutic uses in the suppression of cancer, proliferative diseases and aging.

Dehydroxymethylepoxyquinomicin, a novel nuclear factor-kappaB inhibitor, prevents inflammatory injury induced by interferon-gamma and histamine in NCTC 2544 keratinocytes.

1. The novel nuclear factor (NF)-kappaB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) is a derivative of the antibiotic epoxyquinomicin C from Amycolatopsis sp. that has been found to inhibit tumour necrosis factor (TNF)-alpha-induced activation of NF-kappaB by suppressing nuclear translocation of NF-kappaB. The aim of the present study was to determine the effects of DHMEQ on interferon (IFN)-gamma- and histamine-activated NCTC 2544 keratinocytes. 2. Keratinocytes were stimulated or not with 200 U/mL IFN-gamma and 10(-4) mol/L histamine in the absence or presence of different concentrations of DHMEQ (1, 5 and 10 microg/mL) or hydrocortisone (10(-5) mol/L), which was used as a reference anti-inflammatory drug. After 48 h, each sample was tested for the presence of intercellular adhesion molecule (ICAM)-1 by western blot analysis, as well as for the release of monocyte chemoattractant protein (MCP)-1, RANTES and interleukin (IL)-8 using specific sandwich ELISAs. To verify the effect of DHMEQ on cell viability of non-stimulated NCTC 2544 keratinocytes, the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used. 3. The results showed that 10 microg/mL DHMEQ potently inhibited ICAM-1 production (by 50%), as well as the release of MCP-1 (to 25% of control), RANTES (to 5% of control) and IL-8 (to 2% of control). The results of the MTT assay indicated that DHMEQ has no effect on cell viability. 4. In conclusion, DHMEQ inhibits the IFN-gamma- and histamine-induced activation of the keratinocyte cell line NCTC 2544. The anti-inflammatory effects of DHMEQ could be exploited by applying the drug topically alone or in combination with sub-toxic concentrations of anti-inflammatory drugs to producer a synergistic effect.

State of the art in antigen retrieval for immunohistochemistry.

The masking effects of antigens by chemical fixation, processing, embedding media interactions, represent a serious problem for immunohistochemical purposes. Fortunately, different approaches in antigen retrieval exist. These techniques are relatively recent and continuously expanding. This review focuses on the present state of the art in antigen retrieval methods for immunohistochemistry in light and electron microscopy. Moreover, a brief discussion on the chemical aspects of fixation, mechanism of retrieval, as well as its efficacy, is given.

Uterine cervical carcinoma: role of matrix metalloproteinases (review).

Epidemiological and experimental studies have provided evidence that human papillomavirus (HPV) infection is a main player in the development of uterine cervical neoplasms. Migration of cancer cells from the origin tissue to surrounding or distant organs is essential for tumor progression. Many studies of tumor invasion and metastases have focused on the degradation of the extracellular matrix where matrix metalloproteinases (MMPs) play a central role. Two of these enzymes, MMP-2 and MMP-9, have been correlated with the processes of tumor cell invasion and metastasis in human cancers, including uterine neoplasms. It has been shown that the up-regulation of MMPs is associated with progression of cervical uterine neoplasms. This review describes the current understanding of MMP-2 and MMP-9 expression and activity in pre-cancer and cancer lesions of cervical uterine, which may open new strategies for diagnostic and therapeutic interventions.

Melanoma: molecular pathogenesis and emerging target therapies (Review).

Malignant melanoma is an aggressive tumor of the skin with a poor prognosis for patients with advanced disease. It is resistant to current therapeutic approaches. In melanoma, both the Ras/Raf/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signalling pathways are constitutively activated through multiple mechanisms. Mutations of BRAF have been proposed to contribute to melanoma development. Increased activity of the MAPK pathway prevents apoptosis and induces cell cycle progression. PTEN deletion results in Akt activation. Akt activation can result in the phosphorylation and inactivation of Raf. This decrease in downstream MEK and ERK activation may lead to loss of differentiation or senescence. This review summarizes the most relevant studies focused on the signalling pathways involved in melanomagenesis. New therapeutic strategies are also reported.

Emerging Raf inhibitors.

BACKGROUND: The Raf/MAPK kinase/extracellular-signal-regulated kinase pathway is often activated by genetic alterations in upstream signaling molecules. An integral component of this pathway, BRAF, is also activated by mutation, especially in melanoma and thyroid cancers. The Raf/MAPK kinase/extracellular-signal-regulated kinase pathway has profound effects on proliferative, apoptotic and differentiation pathways as well as the sensitivity and resistance to chemotherapeutic drugs. OBJECTIVES/METHODS: This review discusses targeting of Raf which could control abnormal proliferation in cancer and other proliferative diseases. The important roles that genetics plays in the response of patients to Raf inhibitors is also evaluated. We also discuss the rationales for approaches combining Raf inhibitors and chemotherapeutic drugs. RESULTS/CONCLUSIONS: Various Raf inhibitors have been developed and are being clinically used to treat patients with melanoma, thyroid, hepatocellular and renal cell cancers. Some 'Raf-kinase inhibitors' affect other kinases which are also expressed on malignant cells; yet, these inhibitors have proven useful in the therapy of certain cancer patients. Other more recently developed Raf specific inhibitors have shown success in the treatment of tumors bearing Raf mutations. The development of Raf inhibitors has significantly advanced cancer therapy in the past decade.

Activation of the osteopontin/matrix metalloproteinase-9 pathway correlates with prostate cancer progression.

PURPOSE: Prostate cancer remains the second most frequent cause of tumor-related deaths in the Western world. Additional markers for the identification of prostate cancer development and progression are needed. Osteopontin (OPN), which activates matrix metalloproteinases (MMP), is considered a prognostic biomarker in several cancers. "In silico" and experimental approaches were used to determine whether OPN-mediated MMP activation may be a signal of prostate cancer progression. EXPERIMENTAL DESIGN: Pearson correlation coefficients were computed for each OPN/MMP pair across seven publicly available prostate cancer gene expression data sets. Using Gene Set Enrichment Analysis, 101 cancer-related gene sets were analyzed for association with OPN and MMP-9 expression. OPN, MMP-9, MMP-2 tissue inhibitor of metalloproteinase-1 plasma levels, and MMP gelatinase activity were measured by ELISA and zymography in 96 and 92 patients with prostate cancer and benign prostatic hyperplasia, respectively, and 125 age-matched healthy men. RESULTS: Computational analyses identified a significant correlation only between MMP-9 and OPN, and showed significant enrichment scores in "cell proliferation", "genes constituting the phosphoinositide-3-kinase predictor", "proliferation signature", and "tumor metastasis" gene sets in association with both OPN and MMP-9. Plasma analyses revealed a significant increase in OPN and MMP-9 levels and activity in patients with prostate cancer in association with clinical variables (prostate-specific antigen > 4 ng/mL and Gleason score > 7). Significant correlation between OPN and MMP-9 levels were also observed. Mean plasma levels of OPN and MMP-9 decreased in patients with prostate cancer within 6 months after prostatectomy. CONCLUSIONS: The concordant computational and experimental data indicate that the extent of OPN pathway activation correlates with prostate cancer progression.

Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance.

Growth factors and mitogens use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their receptors to regulate gene expression and prevent apoptosis. Some components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf). Mutations also occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. Even in the absence of obvious genetic mutations, this pathway has been reported to be activated in over 50% of acute myelogenous leukemia and acute lymphocytic leukemia and is also frequently activated in other cancer types (e.g., breast and prostate cancers). Importantly, this increased expression is associated with a poor prognosis. The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of activated Akt to phosphorylate and inactivate different Rafs. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell lineage specific effects. For example, Raf/MEK/ERK is usually associated with proliferation and drug resistance of hematopoietic cells, while activation of the Raf/MEK/ERK cascade is suppressed in some prostate cancer cell lines which have mutations at PTEN and express high levels of activated Akt. Furthermore the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways also interact with the p53 pathway. Some of these interactions can result in controlling the activity and subcellular localization of Bim, Bak, Bax, Puma and Noxa. Raf/MEK/ERK may promote cell cycle arrest in prostate cells and this may be regulated by p53 as restoration of wild-type p53 in p53 deficient prostate cancer cells results in their enhanced sensitivity to chemotherapeutic drugs and increased expression of Raf/MEK/ERK pathway. Thus in advanced prostate cancer, it may be advantageous to induce Raf/MEK/ERK expression to promote cell cycle arrest, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK induced proliferation and drug resistance. Thus the Raf/MEK/ERK pathway has different effects on growth, prevention of apoptosis, cell cycle arrest and induction of drug resistance in cells of various lineages which may be due to the presence of functional p53 and PTEN and the expression of lineage specific factors.

Inflammatory status in patients with chronic renal failure: the role of PTX3 and pro-inflammatory cytokines.

Increased plasma levels of several acute phase proteins, such as C-reactive protein (CRP), have been documented among different patients with chronic renal failure (CRF). The aim of the present study was to determine whether pentraxin-3 (PTX3) is a reliable marker of inflammation in CRF. Plasma samples and monocytes were taken from 43 patients before and after undergoing haemodialysis (HD), from 45 uraemic patients (UR) without HD treatment and from 25 healthy controls. Plasma and monocyte samples were analyzed by ELISA for levels of PTX3, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6); all of these protein levels were higher in CRF patients with respect to the controls. After HD, plasma PTX3 and cytokine levels increased. Inter- and intra-individual variations in CRP were observed in HD patients, while PTX3 plasma levels were stable. Release of PTX3, TNF-alpha, IL-1beta and IL-6 by unstimulated monocytes from patients, before and after HD, was higher with respect to UR patients and controls. After lipopolysaccharide stimulation, all values were higher in patients before HD than those in UR patients, but lower when compared to those in the controls. In contrast, no changes were observed after HD. A significant correlation among plasma PTX3 versus fibrinogen, TNF-alpha and IL-1beta was observed in HD and UR patients. Collectively, these data suggest that PTX3 protein may represent an additional and stable marker of inflammation in CRF.

Detection of BRAF gene mutation in primary choroidal melanoma tissue.

Numerous BRAF mutations have been detected in melanoma biopsy specimens and cell lines. In contrast, several studies report lack of BRAF mutations in uveal melanoma including primary and metastatic choroidal and ciliary body melanomas. To our knowledge, for the first time, here we report a case of choroidal melanoma harboring the BRAF mutation (V600E). The activation of RAF/MEK/ERK pathway, although independent of BRAF mutation, was reported in uveal melanoma. The presence of V600E mutation indicates that the RAF/MEK/ERK pathway, in addition to cutaneous melanoma progression, may play a role in the choroidal melanoma development.

Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance.

The Ras/Raf/MEK/ERK and PI3K/PTEN/AKT signaling cascades play critical roles in the transmission of signals from growth factor receptors to regulate gene expression and prevent apoptosis. Components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf, PI3K, PTEN, Akt). Also, mutations occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. These pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of elevated activated Akt levels to phosphorylate and inactivate Raf-1. We have investigated the genetic structures and functional roles of these two signaling pathways in the malignant transformation and drug resistance of hematopoietic, breast and prostate cancer cells. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell-lineage-specific effects. Induced Raf expression can abrogate the cytokine dependence of certain hematopoietic cell lines (FDC-P1 and TF-1), a trait associated with tumorigenesis. In contrast, expression of activated PI3K or Akt does not abrogate the cytokine dependence of these hematopoietic cell lines, but does have positive effects on cell survival. However, activated PI3K and Akt can synergize with activated Raf to abrogate the cytokine dependence of another hematopoietic cell line (FL5.12) which is not transformed by activated Raf expression by itself. Activated Raf and Akt also confer a drug-resistant phenotype to these cells. Raf is more associated with proliferation and the prevention of apoptosis while Akt is more associated with the long-term clonogenicity. In breast cancer cells, activated Raf conferred resistance to the chemotherapeutic drugs doxorubicin and paclitaxel. Raf induced the expression of the drug pump Mdr-1 (a.k.a., Pgp) and the Bcl-2 anti-apoptotic protein. Raf did not appear to induce drug resistance by altering p53/p21Cip-1 expression, whose expression is often linked to regulation of cell cycle progression and drug resistance. Deregulation of the PI3K/PTEN/Akt pathway was associated with resistance to doxorubicin and 4-hydroxyl tamoxifen, a chemotherapeutic drug and estrogen receptor antagonist used in breast cancer therapy. In contrast to the drug-resistant breast cancer cells obtained after overexpression of activated Raf, cells expressing activated Akt displayed altered (decreased) levels of p53/p21Cip-1. Deregulated expression of the central phosphatase in the PI3K/PTEN/Akt pathway led to breast cancer drug resistance. Introduction of mutated forms of PTEN, which lacked lipid phosphatase activity, increased the resistance of the MCF-7 cells to doxorubicin, suggesting that these lipid phosphatase deficient PTEN mutants acted as dominant negative mutants to suppress wild-type PTEN activity. Finally, the PI3K/PTEN/Akt pathway appears to be more prominently involved in prostate cancer drug resistance than the Raf/MEK/ERK pathway. Some advanced prostate cancer cells express elevated levels of activated Akt which may suppress Raf activation. Introduction of activated forms of Akt increased the drug resistance of advanced prostate cancer cells. In contrast, introduction of activated forms of Raf did not increase the drug resistance of the prostate cancer cells. In contrast to the results observed in hematopoietic cells, Raf may normally promote differentiation in prostate cells which is suppressed in advanced prostate cancer due to increased expression of activated Akt arising from PTEN mutation. Thus in advanced prostate cancer it may be advantageous to induce Raf expression to promote differentiation, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK-induced proliferation. These signaling and anti-apoptotic pathways can have different effects on growth, prevention of apoptosis and induction of drug resistance in cells of various lineages which may be due to the expression of lineage-specific factors.

Long pentraxin 3: a marker of inflammation in untreated psoriatic patients.

Psoriasis is a common cutaneous disorder characterized by abnormal epidermal differentiation, proliferation and inflammation mediated by dermal infiltrates, such as T cells, neutrophils, dendritic cells and macrophages. There are renewed interest in the role of components of the innate immune system. Cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6, and -1beta involved in pathogenic phenomena in psoriasis are known as inducers of the acute phase response. Among the large group of acute phase reactants, C-reactive protein (CRP) and fibrinogen are of special interest in psoriasis. The PTX-3, a long pentraxin sharing similarities with the classical short proteins. Thus, considering the numerous biological roles of inflammatory cytokines and their relationship with inflammatory markers, such as CRP and fibrinogen we have investigated the role of PTX3 in psoriasis. To this aim PTX3, TNF-alpha, IL-6 and IL-1beta in plasma and in monocytic cultures by enzyme linked immunosorbent assay (ELISA) in 44 patients including severe and mild psoriasis were measured. An increased production of PTX3, both in supernatant of purified monocytes and in plasma from patients with severe psoriasis, was found. The significant correlation, between cellular production and plasma levels of PTX3 in psoriasis was found as a sign of cellular activation by monocytes/macrophages that first infiltrate the psoriatic lesion. In severe psoriasis, a significant correlation between psoriasis area and severity index (PASI) score and TNF-alpha and IL-6 levels in both supernatant of monocytes and plasma was found. In contrast, no correlation was found for IL-1beta. By immunohistochemistry and immunofluorescence, a strong PTX3 staining in fibroblasts, endothelial cells and monocytes/macrophages in severe psoriatic lesional skin was detected. Finally, a positive correlation between PTX3 and disease activity of psoriasis was observed as PASI score was elevated. These findings suggest that PTX3 could be used as a further marker of disease activity of psoriasis.

Genotoxic stress leads to centrosome amplification in breast cancer cell lines that have an inactive G1/S cell cycle checkpoint.

Centrosome amplification plays a key role in the origin of chromosomal instability during cancer development and progression. In this study, breast cancer cell lines with different p53 backgrounds were used to investigate the relationship between genotoxic stress, G(1)/S cell cycle checkpoint integrity, and the development of centrosome amplification. Introduction of DNA damage in the MCF-7 cell line by treatment with hydroxyurea (HU) or daunorubicin (DR) resulted in the arrest of both G(1)/S cell cycle progression and centriole duplication. In these cells, which carry functional p53, HU treatment also led to nuclear accumulation of p53 and p21(WAF1), retinoblastoma hypophosphorylation, and downregulation of cyclin A. MCF-7 cells carrying a recombinant dominant-negative p53 mutant (vMCF-7(DNp53)) exhibited a shortened G(1) phase of the cell cycle and retained a normal centrosome phenotype. However, these cells developed amplified centrosomes following HU treatment. The MDA-MB 231 cell line, which carries mutant p53 at both alleles, showed amplified centrosomes at the outset, and developed a hyperamplified centrosome phenotype following HU treatment. In cells carrying defective p53, the development of centrosome amplification also occurred following treatment with another DNA damaging agent, DR. Taken together, these findings demonstrate that loss of p53 function alone is not sufficient to drive centrosome amplification, but plays a critical role in this process following DNA damage through abrogation of the G(1)/S cell cycle checkpoint. Furthermore, these studies have important clinical implications because they suggest that breast cancers with compromised p53 function may develop centrosome amplification and consequent chromosomal instability following treatment with genotoxic anticancer drugs.

Elevated Serum Levels of Osteopontin in HCV-Associated Lymphoproliferative Disorders.

Hepatitis C virus (HCV) infection is associated with chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Recent evidences have also suggested that HCV infection contributes to development of autoimmune disorders and B-cell nonHodgkin's lymphoma (NHL). Mechanisms by which HCV infection promotes B-cell NHL development remain unclear. Increased serum osteopontin (OPN) levels have been associated with several autoimmune diseases as well as a variety of cancers. However, the association between OPN and B-cell NHL or HCV-associated B-cell proliferation has not previously been reported. In the present study, we determined whether serum OPN differences were associated with HCV infection, type II mixed cryglobulinemia (MC) syndrome and B-cell NHL. Serum OPN levels were measured by capture enzyme-linked immunosorbent assay. Our results show that high serum OPN levels are associated with B-cell NHL and HCV infection. Interestingly, highest serum OPN concentrations were found among HCV-infected patients with concomitant type II MC syndrome with and without B-cell NHL. These data indicate that OPN is involved in the lymphomagenesis, especially, in the context of HCV infection and autoimmune diseases.

Aggressive forms of non-Hodgkin's lymphoma in two patients bearing coinfection of Epstein-Barr and hepatitis C viruses.

Although epidemiologic and experimental data suggest an etiopathogenetic role for both hepatitis C virus (HCV) and Epstein-Barr virus (EBV) infection in development of B-cell non-Hodgkin's lymphoma (NHL), potential interactions between EBV and HCV during progression of B-cell NHL have not yet been fully investigated. In the present study, tumor biopsy specimens from patients with both B-cell NHL and chronic HCV infection (HCV(+)) were analyzed for the presence of EBV-encoded RNA (EBER) by in situ hybridization (ISH). VH and VL gene segments were amplified from tumor biopsy specimen DNA by PCR. EBV infection (EBV(+)) was detected in tumors from 2 of 31 (6%) HCV(+) B-cell NHL patients. Clinical histories of these two EBV(+)/HCV(+) B-cell NHL patients indicated a particularly aggressive course of disease. Chemotherapy failed to induce long lasting remission for either of these EBV(+)/HCV(+) B-cell NHL patients. Amplification of CDR3 of the Ig heavy chain gene from DNA isolated from each EBV(+)/HCV(+) B-cell NHL indicated the presence of monoclonal B-cell expansion. Rearrangement of Ig genes in neoplastic B-cell clones from both EBV(+)/HCV(+) patients was similar to that previously reported for EBV(-)/HCV(+) B-cell NHL patients. Additionally, neoplastic B-cell clones from these two EBV(+)/HCV(+) B-cell NHL patients did not exhibit intraclonal variation. Previous studies have demonstrated that intraclonal variation is common among neoplastic B-cell clones from EBV(-)/HCV(+) patients. EBV infection may have prevented evolution of variant neoplastic B-cell clones by suppressing antibody affinity maturation. Together, these data suggest that EBV infection may cooperate with HCV infection during progression of B-cell NHL in immunocompetent individuals. Such an interaction may accelerate the course of disease in B-cell NHL patients.

Bovine seminal ribonuclease is cytotoxic for both malignant and normal telomerase-positive cells.

Bovine seminal-ribonuclease (BS-RNase) is a member of the 'ribonucleases with special biological actions' family since it possesses specific anti-tumour, anti-spermatogenic and embryotoxic activities and exerts an immunosuppressive effect on T lymphocytes. In previous studies it was demonstrated that BS-RNase induced apoptosis in proliferating, malignant and normal cells and that telomerase activity loss also caused apoptotic death in neoplastic cells. Since an obvious relationship between cell proliferation and telomerase activity exists, the aim of this work was to study if the pro-apoptotic cytotoxic action exerted by BS-RNase on proliferating malignant cells (HT29) and proliferating normal cells (PHA-stimulated lymphocytes) could be linked to a possible BS-RNase effect on telomerase activity. In BS-RNase-treated HT29 cells (Na-butyrate-differentiated or not) and human lymphocytes (proliferating or not), we investigated cell vitality (MTT method) and morphology (SEM), BS-RNase localization (immunofluorescence), telomerase activity (TRAP-ELISA method), hTR mRNA expression (RT-PCR), and hTERT levels (western blot). While no BS-RNase effect was detectable on not proliferating cells, a clear relationship was noticed between the diminished number of vital elements of both proliferating cell populations after treatment (48 h and 72 h for HT29 and PHA-stimulated lymphocytes, respectively) with 50 microg/ml BS-RNase and the decrease of their telomerase activity. At the same time, we found that hTR levels, the RNA subunit of telomerase, in proliferation-inhibited BS-RNase-treated cells were diminished. Moreover, by immunofluorescence technique, we detected BS-RNase in the HT29 cell nucleolus after 3-h treatment. Therefore, as hTR has been recently proven to co-fractionate with nucleoli, we hypothesize that a BS-RNase direct action on the telomerase hTR subunit could be a possible mechanism of action by which BS-RNase exerts its pro-apoptotic effects only on proliferating cells.

Cisplatin may be a valid alternative approach in ovarian carcinoma with carboplatin hypersensitivity. Report of three cases.

Platinum-based therapy is considered the standard treatment for patients with advanced ovarian cancer. Carboplatin has a more favorable toxicity profile than cisplatin; however, hypersensitivity reactions to carboplatin have been reported occasionally. We reviewed 112 cases of ovarian cancer treated with carboplatin at our institute to identify the hypersensitivity reactions to this chemotherapeutic agent. Hypersensitivity reactions were documented in nine cases (8%). No deaths occurred, but the reactions were judged severe in three of the 112 patients (2.6%). In our own experience with patients showing idiosyncrasy to carboplatin we observed successful resolution after retreatment with cisplatin. Since patients who relapse after initial treatment with carboplatin often respond to it a second time, it is important to continue this treatment.