Primary Malignant Cells Research Articles

Constitutive heterochromatin DNA fragments are demethylated and decondensed in senescent primary fibroblasts MRC5 and malignant A431 cell line

It is commonly accepted that satellite DNA (satDNA) is highly condensed in the interphase. We checked localization, the degree of condensation, and methylation level of centromeric (CEN) and pericentromeric (periCEN) satDNA fragments by immunofluorescent in situ hybridization (immuno-FISH). An antibody against 5-methylcytosine was used for the immunostaining, and satDNA probes were used for FISH. Cells from the normal somatic tissues (placenta cells and lymphocytes), a primary fibroblast cell line (MRC5), and a malignant cell line (A431) were analyzed. CEN satDNA was condensed and highly methylated in all studied cell types. PeriCEN human satellite 3 from chromosome 1 (HS3-1) was condensed in lymphocytes, placenta cells, and in young cells of the primary culture. In senescent fibroblasts and in the malignant cell line A431, the unfolded HS3-1 was observed. An antibody against methylated DNA stained compact patches of the periCEN satDNA and did not stain the unfolded regions. Thus, we observed the unfolding of the HS3-1 in senescent MRC5 and malignant A431. The unfolding was accompanied by partial demethylation of the satDNA that belongs to the constitutive heterochromatin.

Unusual scapular metastasis as initial manifestation of advanced nonseminomatous germ cell tumor of the mediastinum

Primary malignant germ cell tumors of the mediastinum are relatively rare, occurring predominantly in young male adults, and have a poor prognosis. We present a case of a 27-year-old man who initially experienced a persistent, intractable painful sensation over the right lower scapula despite taking an analgesic agent for 2 months. A scapular x-ray film and a whole-body bone scan showed an expansile osteolytic lesion. Excisional biopsy of the scapula revealed a metastatic carcinoma, suggestive of nonseminomatous germ cell tumor origin. Further examination of the whole abdomen and bilateral testes were negative. Chest computed tomography and magnetic resonance imaging showed a primary tumor mass in the anterior mediastinum. Chemotherapy with cisplatin, bleomycin, and etoposide was administered for six courses. The mediastinal tumor mass was markedly reduced in size and remission without evidence of tracer uptake by [(18)F]fluorodeoxyglucose positron emission tomography examination. Six months after chemotherapy, the patient received advanced surgical intervention to remove the mediastinal tumor, the pathologic features of which were similar to the previous scapular lesion. He was doing well at 1-year follow-up.

The Novel HDAC Inhibitor UCL67022 Is Highly Potent in Multiple Myeloma and Non-Hodgkin's Lymphoma and Is Enhanced by Bortezomib.

Introduction: Histone deacetylases (HDACs) regulate the acetylation state of nucleosomal histones and represent a novel target in haematological malignancies. Inhibition of HDACs results in the induction of apoptosis in multiple myeloma (MM) and lymphoma (NHL) cells, associated with the down-regulation of signaling pathways involving IL-6 and IGF-1 and the inactivation of BCL-6. We have therefore investigated the activity of a novel hydroxamic acid HDAC inhibitor (UCL67022) in comparsion with SAHA, alone and in combination with the proteasome inhibitor bortezomib.Methods: HDAC activity in partially purified rat liver homogenate and intact CEM cells was determined using a fluorescent HDAC substrate. The human MM cell lines RPMI 8226/S and U266, and the NHL cell lines SUD-4, CRL, DOHH2, DHL-4, 5, 6, 7, GRANTA-519 and JEKO-1 were used to investigate effects on viable cell number using an ATP-dependent bioluminescence method. Activity against primary malignant cells from patients with MM, DLBCL, FL or CLL was also studied. Western blot analysis was used to investigate changes in acetylated histone-H3 and a-tubulin.Results:UCL67022 showed more potent HDAC inhibitory activity in liver extracts and whole CEM cells than SAHA (IC50 0.05 vs 0.39uM in liver and 0.11 vs 0.33uM in CEM cells). UCL67022 was more potent than SAHA in reducing viable cell number in U266 (EC50 0.14 vs 1.8 uM) and RPMI 8226/S (0.05 vs 0.78 uM) cells. Similarly NHL cell lines were 10–20 fold more sensitive to UCL67022, with median EC50 values of 0.05uM (range 0.03–0.10uM) vs 0.81uM, (range 0.68–1.28uM). In 2 primary MM samples UCL67022 showed increased activity with EC50 values of 0.7uM and 0.11uM vs 12.2uM and 1.1uM for SAHA. 3 FL patient samples and 1 CLL sample were 3.7-fold more sensitive to UCL67022 than SAHA (median EC50 4.7uM vs 17.5uM respectively). Western blot analysis showed a 10-fold difference in histone H3 acetylation between the two compounds, with acetylation returning to pre-treatment levels by 24hr with 3uM SAHA but remaining elevated out to 48hr with 0.3uM UCL67022. Increased acetylation of a-tubulin confirmed the inhibition of HDAC6. Combination with bortezomib at 1, 2 and 4nM in MM cells showed increased antiproliferative activity with both SAHA and UCL67022, with synergistic responses observed in the U226 cell line and in 1 primary sample and additive effects in the others.Conclusions: These data demonstrate the activity of the highly potent, novel HDAC inhibitor UCL67022 in MM and NHL cell lines and primary patient cells. Activity was increased in co-exposures with bortezomib possibly due to inhibition of HDAC6 and subsequent aggresome formation, suggesting a therapeutic advantage with the combination.

Differential Regulation of D-Type Cyclins by Insulin-Like Growth Factor-I and Serum in Multiple Myeloma Cells.

Dysregulation of a D-type cyclin gene is an early and universal event in multiple myeloma (MM), but given the low proliferative activity in this disease, the functional significance of these genetic lesions is unclear. In this study we first examined the expression and regulation of D-type cyclins and other cell-cycle regulatory proteins in a panel of human myeloma cell lines (HMCLs) and in primary normal and malignant CD138+ plasma cells by Western blotting. D-type cyclins, cyclin dependent kinase-4 (CDK4), CDK6, p27, the retinoblastoma protein (pRb) and proliferating cell nuclear antigen (PCNA) were absent in normal bone marow (BM) CD138+ cells (n=5) and heterogeneously expressed in both HMCLs (n=11) and in primary CD138+ MM cells (n=20; 16 from BM aspirates and 4 extra-medullary). Furthermore, expression of these proteins was positively associated with disease progression. Nine of the primary malignant samples were, like normal CD138+ cells, negative for D-type cyclins, their associated kinases or pRB. Of these, one was from a newly diagnosed patient, six were from patients with stable disease and two from patients on treatment. The remaining eleven MM patient samples had heterogeneous expression of D-type cyclins and other cell cycle regulators, four of which had detectable CDK 4/6-phosphorylated pRb. All four of these were from patients with progressive disease, and three were extra-medullary. Furthermore, we demonstrate for the first time in both HMCLs and primary CD138+ MM cells, that cyclins D1 and -D2 are positively regulated by IGF-I and foetal calf serum leading to increased phosphorylation of pRb on CDK4/6 specific sites and an increase in cells in S (DNA synthesis) phase of the cell cycle. In addition, p27 was down-regulated by IGF-I and FCS in some HMCLs suggesting that this cyclin dependent kinase inhibitor also contributes to cell cycle regulation in myeloma cells. However, at low stoichiometric concentrations observed in primary malignant plasma cells, p27 was up-regulated by mitogenic stimuli, consistent with its role in stabilising cyclin D/CDK complexes. Immunoprecipitation analysis revealed that cyclins D1 and -D2 were present in complexes with both CDKs −4 and −6, suggesting that both of these kinases mediate the effect of cyclin D1 or cyclin D2 on pRb phosphorylation and cell cycle entry in these cells. Unlike HMCLs in which cyclin D2 was the primary controller of cell cycle exit/entry (MM1-S and NCI-H929), HMCLs harbouring an 11q13 IgH translocation (KMS12-BM and U266) were refractory to serum withdrawal, suggesting that 11q13 may confer growth factor independent expression of cyclin D1. To determine if cyclin D1 expression via 11q13 was sufficient to promote cell cycle progression, we functionally inactivated it using siRNA in KMS12-BM. Knock-down of cyclin D1 coincided with decreased CDK4/6-specific pRb phosphorylation and an increase in cells arrested in G1 phase of the cell cycle, confirming that cyclin D1 expression via 11q13 leads to cell cycle progression in MM cells, independent of exogenous growth factors. In summary, we show that D-type cyclins are functional in MM, differentially responsive to exogenous growth factors and that their expression is positively associated with aggressive disease.

Identification of HLA-A*0201-Presented T Cell Epitopes Derived from the Oncofetal Antigen-Immature Laminin Receptor Protein in Patients with Hematological Malignancies

The oncofetal Ag immature laminin receptor (OFA-iLR) is a potential target molecule for immunotherapeutic studies in several tumor entities, including hematological malignancies. In the present study, we characterize two HLA-A*0201-presented epitopes eliciting strong OFA-iLR peptide-specific human cytotoxic T cell (CTLs) responses in vitro. Both allogeneic HLA-A*0201-matched and autologous CTLs recognized and killed endogenously OFA-iLR-expressing tumor cell lines and primary malignant cells from patients with hemopoietic malignancies in an MHC-restricted fashion but spared nonmalignant hemopoietic cells. Spontaneous OFA-iLR peptide-specific T cell reactivity was detectable in a significant proportion of leukemia patients. Interestingly, in patients with chronic lymphocytic leukemia and multiple myeloma but not in those with acute myeloid leukemia, significant frequencies of OFA peptide-specific CTLs could be detected in an early stage of disease but disappeared in patients with progressive disease. The identification of OFA-iLR-derived peptide epitopes provides a basis for tumor immunological studies and therapeutic vaccination strategies in patients with OFA-iLR-expressing malignancies.

In vitro and in vivo reversal of MDR1-mediated multidrug resistance by KT-5720: Implications on hematological malignancies

Multidrug resistance (MDR) due to over-expression of the MDR1 (ABCB1) gene and its P-glycoprotein (Pgp) product is an obstacle in the treatment of hematological malignancies. In this study, we have evaluated the potency of KT-5720 to reverse Pgp-dependent MDR in vitro and in vivo. KT-5720 (but not its close derivatives, K252a and K252b) reversed multidrug resistance of LM1/MDR cell line at non-toxic concentrations and increased accumulation of rhodamine 123 (Rh123). KT-5720 significantly reversed MDR1-dependent resistance of primary malignant cells from patients with chronic myelogenous leukemia in blast crisis (CML-BC) and advanced multiple myeloma (MM). Moreover, KT-5720 (at 5 mg/kg) sensitized the bone marrow of MDR1 transgenic mice model towards daunorubicin (at 8 mg/kg) without general toxic effects. Therefore, KT-5720 can be considered as candidate for combination therapy in various hematological malignancies where Pgp activity is a major impediment for cure.

A novel bispecific protein (ULBP2-BB4) targeting the NKG2D receptor on natural killer (NK) cells and CD138 activates NK cells and has potent antitumor activity against human multiple myeloma in vitro and in vivo

The inability of the immune system to recognize and kill malignant plasma cells in patients with multiple myeloma (MM) has been attributed in part to the ineffective activation of natural killer (NK) cells. In order to activate and target NK cells to the malignant cells in MM we designed a novel recombinant bispecific protein (ULBP2-BB4). While ULBP2 binds the activating NK receptor NKG2D, the BB4 moiety binds to CD138, which is overexpressed on a variety of malignancies, including MM. ULBP2-BB4 strongly activated primary NK cells as demonstrated by a significant increase in interferon-gamma (IFN-gamma) secretion. In vitro, ULBP2-BB4 enhanced the NK-mediated lysis of 2 CD138+ human MM cell lines, U-266 and RPMI-8226, and of primary malignant plasma cells in the allogenic and autologous setting. Moreover, in a nude mouse model with subcutaneously growing RPMI-8226 cells, the cotherapy with ULBP-BB4 and human peripheral blood lymphocytes abrogated the tumor growth. These data suggest potential clinical use of this novel construct in patients with MM. The use of recombinant NK receptor ligands that target NK cells to tumor cells might offer new approaches for other malignancies provided a tumor antigen-specific antibody is available.

Discordant influence of amphotericin B on epirubicin cytotoxicity in primary hepatic malignant cells collected by a new primary culture technique

The purpose of this prospective study was to investigate whether amphotericin B (AmB) had any potential role in the systemic chemotherapy of primary hepatic malignancy using cancer cells collected by the authors' method of primary culture. The specimens obtained by ultrasound-guided fine-needle aspiration biopsy (22 G) from 15 patients with hepatocellular carcinoma (HCC) and one with cholangiocarcinoma were plated into culture flask without disaggregation by trypsin-ethylenediamine tetra-acetic acid solution. Six patients with HCC and one patient with cholangiocarcinoma (7/16, 44%) had successful culture and the cancer cells at the 4th passage were continuously exposed to therapeutic ranges of epirubicin (0, 0.5, 1.0, 1.5, 2.0 microg/mL) with or without the combination of 2.5 microg/mL AmB for 24 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to evaluate the effects of the drugs. A human HCC cell line (HA 22T/VGH) was studied for comparison. Addition of AmB showed no influence on epirubicin cytotoxicity in two patients (one partial resistant HCC and one epirubicin-sensitive cholangiocarcinoma; 25%), augmentation of the epirubicin cytotoxicity in two patients (one total resistant HCC, partial resistant HA 22T/VGH cell line and one epirubicin-sensitive HCC; 37.5%), and decrease of epirubicin cytotoxicity in the remaining three (one partial resistant and two epirubicin-sensitive HCC; 37.5%). Amphotericin B has a discordant influence on epirubicin cytotoxicity in primary cultured hepatic malignant cells. Application of AmB in the systemic chemotherapy of primary hepatic malignancy should be limited to patients with positive AmB effect evaluated by an in vitro sensitivity test such as the present method.

Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents.

ABT-737, an Inhibitor of Bcl-2/Bcl-XL Proteins, Is Cytotoxic in Multiple Myeloma Cells.

Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation.Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA)Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail.Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

Changes in Mcl-1 and Bim Expression with Bortezomib and Melphalan Therapy for Multiple Myeloma.

Background: Proteasome inhibition has been shown to be effective against a variety of tumours. In multiple myeloma the response rates to bortezomib (B) in relapsed patients is 46% (APEX study ≥ MR), but are likely to be up to 75% when combined with melphalan (M). Mechanisms underlying this effect are yet to be fully determined.Aims: To investigate the effect of B, M and the combination of the two on myeloma cell lines and primary patient cells, focusing on the anti-apoptotic molecule Mcl-1 and the pro-apoptotic molecule Bim in the mediation of drug activity. Cell cycle analysis using propidium iodide and flow cytometry was also performed in parallel.Methods: The human multiple myeloma cell lines (HMCLs) RPMI 8226/S, U266 and purified primary patient malignant plasma cells were used for cell culture and viability assays using an ATP bioluminescence method. Cells were incubated for 24 or 48 hours with differing concentrations of B, M and combinations in varying schedules. EC50 values were calculated using a sigmoidal Emax model and the observed cell viability of the combination of the two drugs was compared with the additive effect expected. Mcl-1, Bim, caspase-3, and PARP were probed for by Western Blotting of HMCLs.Results: Both U266 and RPMI 8226/S cells showed dramatic reductions in cell viability to B, with EC50 values of 4.7nM and 5.3nM respectively, and responded to high concentrations of M with EC50 values of 95.6 uM and 91.5 uM respectively after 48 hour incubations. Synergistic responses were seen when M was added 24 hours prior to B, but not with B pre-treatment. This was also observed with primary patient cells.Mcl-1 levels increased after 6 hours of B exposure, likely due to proteasome inhibition, but decreased by 24 hours with associated cleavage. This effect was concentration-dependent with partial cleavage observed at 4nM (approximately EC30) and full cleavage at 50nM. Bim was present in untreated cells, unchanged after 6 hours of B exposure, but decreased at 24 hours at both concentrations. All of these changes were associated with cleavage of caspase-3 and the appearance of cleaved PARP, and persisted out to 48 hours exposure.Six hours following M exposure, there was an increase in Mcl-1 at the sub-toxic 10uM concentration (possibly a cell survival response), but a reduction at 100uM. Following 24/48 hour exposures changes were no different to control cells with 10uM M, however at 100uM (EC50 concentration) cleavage of Mcl-1 and a decrease in Bim were observed, similar to changes seen with 50nM B. On combining the two drugs simultaneously in a 48 hour exposure, B 4nM and M 10uM failed to induce any changes in U266 cells, but resulted in partial cleavage of Mcl-1 in RPMI 8226 cells. When the M concentration was increased to 100uM there was a decrease of both Mcl-1 and Bim and the associated cleavage of caspase-3 and PARP. There were no differences whether B preceded or followed M.Cell cycle analysis demonstrated G2 arrest following B therapy at 24 hours and in combination with M.Conclusions: This work demonstrates that in multiple myeloma, both Mcl-1 and Bim are closely involved in proteasome mediated cellular apoptosis and in M mediated cytotoxicity. In keeping with work in Jurkat cells (Nencioni et al., Blood 2005), Mcl-1 was found to transiently increase following proteasome inhibition, but then decreased at 24 hours as apoptosis occurred. An early rise in anti-apoptotic proteins such as Mcl-1 may explain why synergistic responses with B and M were seen only with M pretreatment.

Activity Profiling of Proteasome Subunits and Deubiquitinating Enzymes in Human Hematopoetic Malignancies.

Proteasomal proteolysis is based on the activity of six different catalytic proteasomal subunits (β1, β2, β5, β1i, β2i, β5i) as well as ubiquitin-mediated recruitment of cytosolic proteins to the proteasomal complex. The latter is regulated by a balance between ubiquitinating enzymes and a variety of ubiquitin-specific proteases (USP). Although proteasomal proteolysis has become a major therapeutic target, the individual activity profiles of proteasomal subunits and USP in primary human malignant cells are unknown. Recently, an activity-based, cell-permeable affinity probe has been developed, which allows to visualize the individual proteasomal subunit activity in living cells in a semiquantitative fashion (Berkers et al., Nature Methods, May 2005). Similarly, a ubiquitin-based synthetic probe (HAUbVS) allows to visualize a broad range of USP based on their activity in cellular lysates. We have used both types of probes to assess i) the differential activity of the proteasomal catalytic subunits in intact cells and ii) the activity profiles of a panel of USP in primary cells of human hematologic malignancies.Individual subunits of both the constitutive and the immuoproteasome were tagged by the probe in live cells in situ, resolved by 1D or 2D SDS-PAGE, and identified by mass spectrometry. Comparison of different cases of ALL and CLL revealed a remarkable variability in the relative activities of the β1/1i–, β2/2i–, and β5/5i–type of subunits, respectively, which contrasted with their stochiometrically constant relation of the respective polypeptides in the architecture of the proteasome. Of note, live primary myeloma cells lacked active β1/1i–type subunits and contained only extremely low amounts of β5/5i–type active subunits, while active β2/2i subunits were present in amounts comparable with primary leukaemia cells or monocytes. The variability in active proteasomal subunits contrasted with a relatively homogeneous distribution of the activity profiles of seven different USP that were targeted by the USP-specific probe. In particular, and in contrast to control cell lines, we failed to confirm the substantial upregulation of UCH-L1 in primary cells, which had been suggested to be a characteristic of the proteasomal machinery in malignant cells, based on studies using human cell lines.In addition, we here for the first time directly visualize the effect of bortezomib on individual proteasome subunits in primary human cells: although Bortezomib is reported to selectively target the β5 subunit, we here show that both the β1 and β1i subunits are also inhibited by the substance, the latter with even higher affinity (IC50 approx. 2.5 nM). In one case of ALL that lacked detectable active β5/5i subunits in intact cells, Bortezomib induced cytotoxic death at the concentration where β1/β1i activity was eliminated.In summary, our study for the first time visualizes the differential activity of proteasomal subunits in living primary human cells, demonstrates significant variability in their activity profiles in leukaemia and myeloma cells and suggests that differential proteasomal activity might contribute to the variable sensitivity of cancer cells towards proteasome inhibitors such as Bortezomib.

Small-cell variant of renal oncocytoma with dominating solid growth pattern: a potential diagnostic pitfall

Dear Editor, Renal oncocytoma is a distinct benign renal tumour accounting approximately for 4% of all renal tumours. This neoplasm is easily recognizable in its classic form— polygonal cells with abundant granular eosinophilic cytoplasm, arranged in tubuloalveolar cystic pattern— whereas diagnostic difficulties may arise when it exhibits small cells with scant cytoplasm and hyperchromatic nuclei (so-called “oncoblasts”) [2]. Although these small cells are rarely detectable in an otherwise classic renal oncocytoma [1, 3], there is the possibility that they may represent the dominating cell component [2]. One of us (MM), by reviewing a large series of renal oncocytomas, found only 3 out of 134 tumours with a prominent (80% of the entire tumours in two cases and 50% in the other case) small-cell component, and the term “small-cell variant of renal oncocytoma” was proposed for these cases [2]. We report a rare case of renal oncocytoma with a predominant small-cell component, accounting for about 80% of the entire tumour. The unusual extensive solid growth pattern of the tumour may represent a potential diagnostic pitfall with primary or metastatic malignant small cell tumours. A 58-year-old woman presented with a vague right-flank pain. Ultrasonography and computerized tomography examination revealed a renal mass, measuring 3.5 cm in greatest dimension and located in the upper pole. A radical nephrectomy with abdominal lymph node dissection was performed. Grossly, a well-circumscribed, nonencapsulated tumour mass, tan in colour, was observed. No central scar or haemorraghic or necrotic areas were seen. Histologically, the tumour was mainly (about 80% of the entire tumour) composed of small cells with scant pale to slightly eosinophilic cytoplasm and small hyperchromatic round-shaped nuclei, arranged in a solid growth pattern (Fig. 1a,b). Only focally, the tumour cells exhibited a tubular and alveolar growth pattern. After an extensive search, in some tumour areas, we observed that neoplastic small cells gradually merged with larger cells with abundant granular eosinophilic cytoplasm similar to oncocytes as seen in classic oncocytoma (Fig. 1c). In other areas, the transition between the small cells and the classic oncocytes was abrupt (Fig. 1d). Only the small foci of tumour were composed exclusively of typical oncocytes (Fig. 1e). Nuclear pleomorphism was focally observed in classical oncocytes, as well as binucleation. Mitoses, necrosis, and nuclear pleomorphism were absent. Perirenal adipose tissue invasion and lymph node metastasis were not seen. The patient is well, without evidence of tumour after a 2-year follow-up. Immunohistochemical analyses revealed a faint immunoreactivity to antimitochondrial antibody MITO-113 (Biogenex) in the small-cell component (Fig. 1f), whereas a stronger staining was observed in typical oncocytes. All neoplastic cells were positive to epithelial membrane antigen and pancytokeratins. No reactivity was obtained with chromogranin A, synaptophysin, CD56, neuron-specific enolase, leucocyte common antigen, CD34 and CD99. Differential diagnosis mainly revolved around malignant small-cell tumours. Among these, metastatic or primary neuroendocrine carcinomas (small cell G. Magro (*) Dipartimento G.F. Ingrassia, Anatomia Patologica, Universita di Catania, Via S. Sofia 87, 95123 Catania, Italy e-mail: g.magro@unict.it Tel.: +39-95-3782024 Fax: +39-95-3782023

Induction of apoptosis by spermine-metabolites in primary human blood cells and various tumor cell lines

Polyamines are involved in the regulation of cellular growth and survival by interacting with processes like translation, transcription or ion transport. The aim of our study was to analyze whether polyamines induce apoptosis in hematopoetic cells and to investigate the molecular mechanisms involved. We found an induction of apoptosis by spermine in primary human cells and malignant tumor cell lines. Spermine-treatment resulted in an intracellular increase of reactive oxygen species. Apoptosis was mediated by a collapse of mitochondrial membrane potential, a decrease in Bcl-2 expression and a release of apoptosis mediating molecules from mitochondrial intermembrane space (cytochrome C, Smac/DIABLO). Spermine-mediated apoptosis was caspase-dependent. To test whether spermine mediates apoptosis through metabolites we analyzed the effects of several molecules that interfere with its catabolism. Aminoguanidine, an inhibitor of serum amine oxidase, aldehyde-dehydrogenase, which degrades aldehydes to less reactive molecules or N-acetyl-cysteine, a glutathion precursor, significantly inhibited spermine-mediated apoptosis. From these data we conclude that spermine-derived aldehydes and intracellular accumulation of reactive oxygen species result in mitochondria mediated apoptosis.

Different Activation of Mitogen-Activated Protein Kinase and Akt Signaling Is Associated with Aggressive Phenotype of Human Meningiomas

Activation of intracellular signaling cascades has been implicated in the growth control of benign meningiomas, but their role for meningioma progression and outcome is unknown. Here we determined the expression and function of proteins involved in mitogen-activated protein kinase (MAPK) and phosphinositol-3 kinase (PI3K)/Akt signaling in benign, atypical, and malignant meningiomas and studied their association with clinicopathologic data including meningioma recurrence. Expression of various MAPK and PI3K signaling proteins was determined in 70 primary meningiomas and, if present, in recurrent tumors by immunohistochemistry and Western blotting. The expression patterns in primary and recurrent tumors were related to clinical data. The effect of MAPK and PI3K pathway inhibition on cell proliferation and apoptosis was determined using a primary malignant meningioma cell culture. Atypical and malignant meningiomas showed higher levels of phospho-Akt compared with benign tumors, and their proliferation could be inhibited by PI3K blocking using wortmannin. PI3K inhibition did not induce apoptosis in malignant meningioma cells. In contrast, expression of phospho-Raf and phospho-MAPK was decreased in aggressive meningiomas compared with benign tumors, but MAPK inhibition by PD98059 resulted in tumor cell apoptosis and decreased proliferation. Reduced MAPK activation was associated with meningioma recurrence, and PI3K activation was associated with poor preclinical condition and brain invasion of malignant meningiomas. Both MAPK and PI3K/Akt pathways are activated at different levels in benign and malignant meningiomas. Activation of PI3K/Akt signaling contributes to the aggressive behavior of malignant meningiomas, whereas MAPK activation is involved in both proliferation and apoptosis of malignant meningiomas.

A Novel Plasmacytoid Dendritic Cell Line, CAL-1, Established from a Patient with Blastic Natural Killer Cell Lymphoma

Blastic natural killer (NK) cell lymphoma corresponding to CD4+CD56+ malignancies is a novel disease entity, according to the results of clinical, morphologic, and immunologic studies. It is especially noteworthy that this disease likely arises from plasmacytoid dendritic cells (pDCs), described previously as plasmacytoid T-cells, which have an important role in innate and adaptive immunity. However, the exact relationship between the tumor cells and pDCs remains to be elucidated. We encountered a patient with typical blastic NK cell lymphoma, which later converted to leukemic manifestations, and tried to establish a cell line using the leukemic cells. We succeeded in establishment of a novel cell line, CAL-1, which originated from the primary malignant cells. The genetic and phenotypic features of CAL-1 cells bear a similarity to those of pDCs, namely, plasmacytoid morphology at light and electron microscopy; negative results for CD11c and lineage-associated markers of CD3, CD14, CD19, and CD16; positive results for HLA-DR, CD4, CD56, CD45RA, and CD123; and negative results for TCR and IgH gene rearrangements. An interesting finding was that CAL-1 cells change morphologically into the mature DC appearance with many long dendrites after short-term culture in the presence of granulocyte-macrophage colony-stimulating factor and interleukin 3. CAL-1 cells can secrete tumor necrosis factor alpha but not interferon alpha. Thus although they do not share in part phenotypic and functional features with their normal counterparts, CAL-1 cells mostly exhibit a striking pDC phenotype. We describe the first novel pDC cell line of CAL-1. This cell line should open the opportunity for study not only of CD4+CD56+ tumor cells but also of pDCs in vitro.

Morphology and immunohistochemical characteristics of hepatic primary and metastatic malignant spindle cell tumors

To investigate the morphology and immunohistochemical characteristics of hepatic primary and metastatic malignant spindle cell tumors, and to conclude the diagnostic and differential diagnostic criteria for these morphologically similar tumors. Forty-six specimens of hepatic spindle cell tumors. 20 primary tumors (43.4%), including 3 cases of sarcomatoid carcinoma (6.5%), 11 of angiosarcoma (23.9%), 2 of epithelioid hemangioendothelioma (5%), 1 of spindle cell carcinoid (2.2%), and 3 of undifferentiated sarcoma (6.5%). and 26 metastatic malignant tumors (56.5%), including 20 cases of gastrointestinal stromal tumors (GIST, 43.4%), 3 of leiomyosarcoma (6.5%), 2 of malignant peripheral never sheath tumor (4.3%), and 1 of meningeal hemangiopericytoma (2.2%), resected during operation or collected during imaging-mediated liver puncture underwent hematoxylin-eosin staining, SP staining, and EnVision immunohistochemical staining. Either primary or metastatic tumors showed extensive overlapping in histopathologic appearance, and hemangiopericytoma-like structure was the predominant pattern, which could be seen in nearly every kind of hepatic spindle cell tumors. The morphology of GIST was the most complex. Two or three entirely different kinds of structure could be seen in one specimen of GIST, especially in the resected ones. Most stromal tumor cases were CD117 positive, and existed the condition that the primary tumor was positive and the metastatic tumor was negative or vice versa or one part of specimen was positive but other part was negative. Leiomyosarcoma was immunoreactive to smooth muscle specific antigen (SMA), malignant peripheral nerve sheath tumor was immunoreactive to S-100 protein and neurofilament (NF), and both were CD117 negative. Angiosarcoma and epithelioid hemangioendothelioma expressed different immunoreactivity to CD31, CD34 and factor VIII related antigen. Sarcomatoid carcinoma was positive for CK and vimentin. Spindle cell carcinoid was positive for CK and neuroendocrine markers, such as synaptophysin and chromogranin A. Primary angiosarcoma is the most common form of primary spindle cell tumor in liver, and metastatic GIST is predominant in hepatic metastatic spindle cell tumors. A penal of immunohistochemical markers is necessary for the final diagnosis of these tumors because of the complexity and similarity of different tumors of this kind.

Identification of 41 Novel Promoter-Associated CpG Islands Methylated in Leukemias.

DNA methylation within promoter-associated CpG islands is a well-recognized mechanism of gene silencing and plays an important role in the development of malignancies. CpG dinucleotides in human DNA are methylated at 5′-cytosine with the exception of areas with dense concentration of CpGs (CpG islands) located in gene promoter regions. In cancer cells, methylation of CpG islands in promoter regions of tumor suppressor genes is a frequent epigenetic change with a gene-silencing effect analogous to inactivating mutations. Methylation profiling can identify biologically and clinically distinct tumor subgroups by mapping the methylation status of multiple genes, and reports in AML and ALL suggest associations between methylation and poor prognosis. Identification of methylated CpG islands can shed new light on the biology of leukemia. We used Methylated CpG Island Amplification coupled with Representative Difference Analysis (MCA-RDA) as a genome-wide screen for promoter-associated CpG islands methylated in leukemic and/or myeloproliferative cell lines and primary malignant cells, but unmethylated in blood cells from normal controls. We identified 51 unique promoter-associated CpG islands in 321 sequenced clones recovered by MCA-RDA. Forty-one CpG islands belonged to known genes, and 10 to annotated mRNAs. Of the genes with known function, 8 are involved in signaling, 7 in transcription, 3 in dephosphorylation, 2 in oxido-reductive processes, 2 in NO synthesis, 2 in adhesion, 2 in solute transport, and 2 in DNA replication. Seven out of the 51 genes were previously reported as methylated in cancer or leukemia (CDH13, HLA-B, HLA-C, PGR, SCGB3A1, SLC26A4, TERT), thus validating the MCA-RDA approach. Of the 41 new hypermethylated CpG islands recovered, 20 corresponded to genes of known function. Published data infer an association with cancer for 10 of these genes (CTDSPL, ECGF1, EDG4, FOXD2, NOR1, NOS3, OLIG2, SLC16A1, TLE1, WNT5B), and no reports were found for the other 10 genes (CNR1, FADS, FBXW3, FGD1, NPM2, P518, PDE4DIP, SNCB, TCEA3, VENTX2). To further validate our findings we are assessing the methylation status of these genes by bisulfite pyrosequencing. Analyses of the bone marrow samples from AML, ALL, CML and MDS patients are ongoing. Our preliminary data confirm methylation of H-cadherin precursor (CDH13), progesterone receptor (PGR) in AML and ALL and cannabinoid receptor 1 (CNR1) in ALL (Table). In conclusion, MCA-RDA identified methylation of 41 new and 10 previously reported promoter-associated CpG islands in leukemia. Functional studies of these may shed new light on the biology of leukemias, and these genes may be useful for methylation profiling of leukemias for prognosis and response to treatment.Promoter CpG Island MethylationGeneAMLALLCDH135/2322%12/1963%PGR A Isoform11/2250%12/1867%PGR B Isoform17/2471%5/1338%CNR10/240%3/1817%Methylation levels over 10% for CDH13 and PGR and over 25% for CNR1 were scored as positive.

Identification and Characterization of Small Molecule Inhibitors of the Calcium-Dependent S100B−p53 Tumor Suppressor Interaction

The binding of S100B to p53 down-regulates wild-type p53 tumor suppressor activity in cancer cells such as malignant melanoma, so a search for small molecules that bind S100B and prevent S100B-p53 complex formation was undertaken. Chemical databases were computationally searched for potential inhibitors of S100B, and 60 compounds were selected for testing on the basis of energy scoring, commercial availability, and chemical similarity clustering. Seven of these compounds bound to S100B as determined by steady state fluorescence spectroscopy (1.0 microM < or = K(D) < or = 120 microM) and five inhibited the growth of primary malignant melanoma cells (C8146A) at comparable concentrations (1.0 microM < or = IC(50) < or = 50 microM). Additionally, saturation transfer difference (STD) NMR experiments confirmed binding and qualitatively identified protons from the small molecule at the small molecule-S100B interface. Heteronuclear single quantum coherence (HSQC) NMR titrations indicate that these compounds interact with the p53 binding site on S100B. An NMR-docked model of one such inhibitor, pentamidine, bound to Ca(2+)-loaded S100B was calculated using intermolecular NOE data between S100B and the drug, and indicates that pentamidine binds into the p53 binding site on S100B defined by helices 3 and 4 and loop 2 (termed the hinge region).

Inhibiting S100B Restores p53 Levels in Primary Malignant Melanoma Cancer Cells

S100 calcium-binding proteins such as S100B are elevated in primary malignant melanoma and are used as markers for this and numerous other cancers. Wild-type p53 protein levels are relatively low in these cancer cells (i.e. when compared with cells without S100B) but are elevated when RNA antisense to S100B is introduced. This result implicates S100B in the down-regulation of p53 and is consistent with the large decreases in p53 protein levels observed previously in transient co-transfections of p53 and S100B (Lin, J., Blake, M., Tang, C., Zimmer, D., Rustandi, R. R., Weber, D. J., and Carrier, F. (2001) J. Biol. Chem. 276, 35037-35041). Down-regulation of p53 in primary malignant melanoma cells is likely the result of a direct interaction with S100B, which was observed by co-immunoprecipitation experiments. Furthermore, p53 binds regions of the S100B promoter, one of which matches the 20-nucleotide p53-binding consensus DNA sequence perfectly. Therefore, when p53 levels increase, it contributes to its own demise by up-regulating the transcription of S100B as part of a negative feedback loop. This is analogous to what is found for another protein that down-regulates p53, namely hdm2 (human double mutant 2).