Clinicopathological characteristics of metaplastic breast cancer - analysis of the basic immunohistochemical profile and comparison with other invasive breast cancer types.

INTRODUCTION: Metaplastic breast cancer (MpBC) is a rare but aggressive type of breast cancer accounting for 0.25-1% of all diagnosed invasive breast cancers. Morphologically, it is characterized by differentiation of the neoplastic epithelium into squamous cells and/or mesenchymal-looking tissue. MATERIAL AND METHODS: We analyzed 13 MpBCs selected from the group of 1122 invasive breast cancers. Histopathological examination and analysis of estrogen (ER), progesterone (PR) and HER2 receptors expression in MpBC patients and their comparison to other types of invasive breast cancer has been performed. RESULTS: 13 MpBC cases represented 1.16% of the 1122 invasive breast cancers. The MpBC group presented with a significantly larger tumor size (≥T2, 69% versus 49%, p < 0.001) and with higher grade of histological malignancy (G1-G3) (p < 0.001). MpBC group had significantly more cases with no hormone receptors (ER, PR) and HER2 overexpression/gene amplification compared with the other invasive breast cancer types group (ER-, 69% versus 23%, p < 0.001; PR-, 69% versus 28%, p < 0.001; HER2 0/1+, 93% versus 82%, p = 0.019). Most MpBCs (62%) were triple-negative. We found a correlation between hormone receptors expression and lymph node metastasis (p < 0.001). The analysis of the HER2 expression allowed us to find correlation between its expression and tumor histological grade (G1-G3) (p < 0.001), tumor size (T1a-T4) (p < 0.001) and lymph node metastasis (pN0-pN4) (p < 0.001) in MpBCs. DISCUSSION: MpBCs are usually larger at primary diagnosis and most of MpBCs present with other poor prognostic indicators and show lack of steroid hormone receptors expression as well as HER2. Hormone receptor status and HER2 expression seems to correlate with histological grade of malignancy (G1-G3), tumor size (T1a-T4) and regional lymph node involvement (pN0-pN4) and these features are directly related to MpBC malignancy.

A multicentre, open-label, phase-I/randomised phase-II study to evaluate safety, pharmacokinetics, and efficacy of nintedanib vs. sorafenib in European patients with advanced hepatocellular carcinoma.

BACKGROUND: This multicentre, open-label, phase-I/randomised phase-II trial evaluated safety, pharmacokinetics, maximum-tolerated-dose (MTD) per dose-limiting toxicities (DLTs), and efficacy of nintedanib vs. sorafenib in European patients with unresectable advanced hepatocellular carcinoma (aHCC). METHODS: Phase I: Patients were stratified into two groups per baseline aminotransferase/alanine aminotransferase and Child-Pugh score; MTD was determined. Phase II: Patients were randomised 2:1 to nintedanib (MTD) or sorafenib (400-mg bid) in 28-day cycles until intolerance or disease progression. Time-to-progression (TTP, primary endpoint), overall survival (OS) and progression-free survival (PFS) were determined. RESULTS: Phase-I: no DLTs observed; nintedanib MTD in both groups was 200 mg bid. Phase-II: patients (N = 93) were randomised to nintedanib (n = 62) or sorafenib (n = 31); TTP was 5.5 vs. 4.6 months (HR = 1.44 [95% CI, 0.81-2.57]), OS was 11.9 vs. 11.4 months (HR = 0.88 [95% CI, 0.52-1.47]), PFS was 5.3 vs. 3.9 months (HR = 1.35 [95% CI, 0.78-2.34]), respectively (all medians). Dose intensity and tolerability favoured nintedanib. Fewer patients on nintedanib (87.1%) vs. sorafenib (96.8%) had drug-related adverse events (AEs) or grade ≥ 3 AEs (67.7% vs. 90.3%), but more patients on nintedanib (28 [45.2%]) had AEs leading to drug discontinuation than did those on sorafenib (7 [22.6%]). CONCLUSIONS: Nintedanib may have similar efficacy to sorafenib in aHCC.

A randomized, double-blind, placebo-controlled phase II study to assess the efficacy and safety of mapatumumab with sorafenib in patients with advanced hepatocellular carcinoma.

BACKGROUND: This randomized, double-blind, placebo-controlled, phase II study evaluated the efficacy and safety of mapatumumab (a human agonistic monoclonal antibody against tumor necrosis factor-related apoptosis-inducing ligand receptor 1) in combination with sorafenib in patients with advanced hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Patients with advanced HCC (stratified by Barcelona Clinic Liver Cancer stage and Eastern Cooperative Oncology Group performance status) were randomized 1:1 to receive sorafenib (400 mg, twice daily per 21-day cycle) and either placebo (placebo-sorafenib arm) or mapatumumab (30 mg/kg on day 1 per 21-day cycle; mapatumumab-sorafenib arm). The primary end point was time to (radiologic) progression (TTP), assessed by blinded independent central review. Key secondary end points included progression-free survival, overall survival, and objective response. RESULTS: In total, 101 patients were randomized (placebo-sorafenib arm: N = 51; mapatumumab-sorafenib arm: N = 50). There was no significant difference in median TTP between both arms [5.6 versus 4.1 months, respectively; adjusted hazard ratio (one-sided 90% confidence interval) 1.192 (0-1.737)]. No mapatumumab-related benefit was identified when TTP was evaluated in the stratified subgroups. The addition of mapatumumab to sorafenib did not demonstrate improvement in the secondary efficacy end points. The reported frequency of adverse events (AEs) and serious AEs was comparable in both treatment arms. CONCLUSIONS: The addition of mapatumumab to sorafenib did not improve TTP or other efficacy end points, nor did it substantially change the toxicity profile of sorafenib in patients with advanced HCC. Based on these results, further development of the combination of mapatumumab and sorafenib in HCC is not planned.

Efficacy and safety of a nanofiltered liquid intravenous immunoglobulin product in patients with primary immunodeficiency and idiopathic thrombocytopenic purpura.

BACKGROUND AND OBJECTIVES: In the production process of a new 5% liquid intravenous immunoglobulin (IVIG-L) product (Nanogam(®) ), a combined pepsin/pH 4·4 treatment/15-nm filtration (pH 4·4/15NF) step and a solvent-detergent (SD) treatment step were incorporated to improve the virus inactivating/reducing capacity of the manufacturing process. Two prospective uncontrolled multicentre studies were performed to evaluate the safety and efficacy of this product. MATERIALS AND METHODS: Efficacy, including pharmacokinetics, of IVIG-L was studied for 6 months in 18 primary immunodeficiency (PID) patients, succeeded by a long-term follow-up study (mean 2·2 years, n=17). Second, in 24 patients with idiopathic thrombocytopenic purpura (ITP), IVIG-L was studied for efficacy for 14 days. In both studies, adverse events and vital signs were recorded to study safety. RESULTS: In PID patients treated with IVIG-L, 0·60 and 0·38 severe infections per patient per year were reported during, respectively, the short-term and long-term follow-up. Pharmacokinetic studies resulted in an IgG half-life of 30·9 ± 11·3 days and a mean IgG trough level of 6·8 ± 1·2 g/l. In the ITP study, all patients showed an increase in platelet counts after infusion with IVIG-L, and 20/24 patients responded with a platelet count >50 × 10(9) /l (83·3%) within 1 week. IVIG-L infusions did not cause clinical relevant changes in laboratory parameters or vital signs. CONCLUSIONS: In clinical studies, IVIG-L (Nanogam®) demonstrated to be efficacious, well tolerated and safe.

Induction of apoptosis in bone marrow cells after treatment of mice with WR-2721 and gamma-rays: relationship to the cell cycle.

Apoptosis and cell proliferation are accepted to be responsible for the maintenance of homeostasis in the hematopoietic system. Understanding of the mechanisms of action of the aminothiols and ionizing radiation on normal hematopoietic cells requires determination of the correlation between apoptotic cell death and cell cycle distribution. The effects of WR-2721 ((S)-2-/3-aminopropylamino/ethylphosphorothioic acid; Amifostine) and 60Co gamma-rays on apoptosis and cell cycle progression in the mouse bone marrow were determined. Adult male Swiss mice were exposed to 6 Gy gamma-rays only, or pretreated with WR-2721, at a dose of 400 mg/kg body weight, 30 min before gamma-irradiation. The laser scanning cytometry APO-BRDU assay based on simultaneous analysis of cellular DNA content and the in situ detection of DNA strand breaks was used to identify apoptotic cells and to reveal the cell cycle position of apoptotic and nonapoptotic cells. Temporary changes in the frequency of apoptotic cells with fluorescein isothiocyanate (FITC) labeling of DNA strand breaks, and all bone marrow cells including apoptotic and nonapoptotic ones, whose DNA stained with propidium iodide, were observed in the particular phases of the cell cycle throughout the 96-h period after WR-2721 application and gamma-irradiation. The cell cycle phase specificity of WR-2721 and 60Co gamma-irradiation was shown in terms of induction of apoptosis in bone marrow cells. The patterns of alterations in the frequency of apoptotic cells and all bone marrow cells with respect to their cell cycle position were dependent on the agent(s) applied and the time interval after treatment of mice with WR-2721 and/or gamma-rays. A modulatory, suppressive action of WR-2721 on apoptosis induction and the cell cycle perturbation caused in normal cells of the mouse bone marrow by gamma-rays was found.

Effects of WR-2721 and cyclophosphamide on the cell cycle phase specificity of apoptosis in mouse bone marrow.

Elucidation of the mechanisms of action of the thiol and alkylating agents on normal cells requires the knowledge of their cell cycle phase specificity in terms of their ability to induce apoptosis. The effects of S-2-/3-aminopropylamino/ethyl phosphorothioic acid (WR-2721, Amifostine) and cyclophosphamide (CP) on apoptosis and cell cycle progression were assessed in the mouse bone marrow. Adult male Swiss mice were treated with WR-2721, at a dose of 400 mg/kg body weight, and/or CP, at a dose of 200 mg/kg body weight. Application of the laser scanning cytometry APO-BRDU assay, a two-color staining method for labeling of DNA breaks and cellular DNA, allowed an identification of apoptotic and non-apoptotic cells, and their position with respect to their cell cycle phase. Temporary alterations in the number of apoptotic cells and also all bone marrow cells, including apoptotic and non-apoptotic ones, were determined throughout the 240-h period after treatment of mice with WR-2721 and/or CP. These drugs, given alone, affected apoptotic cell death and caused deregulation of the cell cycle in the bone marrow. WR-2721, applied 30 min prior to CP administration, resulted in a suppressing effect on apoptosis and the cell cycle perturbation triggered in normal bone marrow cells by the alkylating drug. The patterns of changes in the frequency of apoptotic cells and the number of apoptotic and non-apoptotic bone marrow cells, observed in all phases of the cell cycle, were dependent on the agent(s) given and the time interval after WR-2721 and/or CP administration.

Effects of the flavonoid baicalin and its metabolite baicalein on androgen receptor expression, cell cycle progression and apoptosis of prostate cancer cell lines.

Recent studies on the Chinese herbal medicine PC SPES showed biological activities against prostate cancer in vitro, in vivo and in patients with advanced stages of the disease. In investigating its mode of action, we have isolated a few of the active compounds. Among them, baicalin was the most abundant (about 6%) in the ethanol extract of PC SPES, as determined by HPLC. Baicalin is known to be converted in vivo to baicalein by the cleavage of the glycoside moiety. Therefore, it is useful to compare their activities in vitro. The effects of baicalin and baicalein were studied in androgen-positive and -negative human prostate cancer lines LNCaP and JCA-1, respectively. Inhibition of cell growth by 50% (ED(50)) in LNCaP cells was seen at concentrations of 60.8 +/- 3.2 and 29.8 +/- 2.2 microM baicalin and baicalein, respectively. More potent growth inhibitory effects were observed in androgen-negative JCA-1 cells, for which the ED(50) values for baicalin and baicalein were 46.8 +/- 0.7 and 17.7 +/- 3.4, respectively. Thus, it appears that cell growth inhibition by these flavonoids is independent of androgen receptor status. Both agents (1) caused an apparent accumulation of cells in G(1) at the ED(50) concentration, (2) induced apoptosis at higher concentrations, and (3) decreased expression of the androgen receptor in LNCaP cells.

Telomerase expression and activity are coupled with myocyte proliferation and preservation of telomeric length in the failing heart.

The role and even the existence of myocyte proliferation in the adult heart remain controversial. Documentation of cell cycle regulators, DNA synthesis, and mitotic images has not modified the view that myocardial growth can only occur from hypertrophy of an irreplaceable population of differentiated myocytes. To improve understanding the biology of the heart and obtain supportive evidence of myocyte replication, three indices of cell proliferation were analyzed in dogs affected by a progressive deterioration of cardiac performance and dilated cardiomyopathy. The magnitude of cycling myocytes was evaluated by the expression of Ki67 in nuclei. Ki67 labeling of left ventricular myocytes increased 5-fold, 12-fold, and 17-fold with the onset of moderate and severe ventricular dysfunction and overt failure, respectively. Telomerase activity in vivo is present only in multiplying cells; this enzyme increased 2.4-fold and 3.1-fold in the decompensated heart, preserving telomeric length in myocytes. The contribution of cycling myocytes to telomerase activity was determined by the colocalization of Ki67 and telomerase in myocyte nuclei. More than 50% of Ki67-positive cells expressed telomerase in the overloaded myocardium, suggesting that these myocytes were the morphological counterpart of the biochemical assay of enzyme activity. Moreover, we report that 20--30% of canine myocytes were telomerase competent, and this value was not changed by cardiac failure. In conclusion, the enhanced expression of Ki67 and telomerase activity, in combination with Ki67-telomerase labeling of myocyte nuclei, support the notion that myocyte proliferation contributes to cardiac hypertrophy of the diseased heart.

Unique analytical capabilities of laser scanning cytometry (LSC) that complement flow cytometry.

Flow cytometry has become an indispensable instrumentation in many disciplines of biology and medicine. There are some limitations of flow cytometry, inherent to the fact that the cells are measured in flow, which limit its usefulness in some applications. The microscope-based laser scanning cytometer (LSC) has many features similar to flow cytometry but few restrictions of the latter and therefore it is useful in many new applications. This review briefly outlines the applications that are unique to LSC, particularly related to its morphometric capabilities and the possibility of cell relocation. Potential future applications of LSC are also discussed.

Telomere shortening is an in vivo marker of myocyte replication and aging.

To determine whether adult cardiac myocytes are capable of multiple divisions and whether this form of growth is restricted to a subpopulation of cells that retain this capacity with age, telomere lengths were measured in myocyte nuclei isolated from the left ventricle of fetal and neonatal Fischer 344 rats and rats at 4, 12, and 27 months after birth. Two independent methodologies were used for this analysis: laser scanning cytometer and confocal microscopy. In each case, fluorescence intensity of a peptide nucleic acid probe specific for telomeric sequence was evaluated. The two techniques yielded comparable results. Telomeric shortening increased with age in a subgroup of myocytes that constituted 16% of the entire cell population. In the remaining nondividing cells, progressive accumulation of a senescent associated nuclear protein, p16(INK4), was evidenced. In conclusion, a significant fraction of myocytes divides repeatedly from birth to senescence, counteracting the continuous death of cells in the aging mammalian rat heart.

Clinical and laboratory evaluation of all-trans retinoic acid modulation of chemotherapy in patients with acute myelogenous leukaemia.

All-trans retinoic acid (ATRA) is synergistic with chemotherapy in leukaemia cell lines. We treated 53 patients with newly diagnosed acute myelogenous leukaemia (AML) with high-dose cytarabine-based chemotherapy followed by ATRA. Peripheral blood and bone marrow samples were obtained to study the effect of in vitro exposure to ATRA and to measure apoptosis and bcl-2. The response rate was 72% for patients under age 60 years and 46% for patients aged 60 years or above. There was no difference in the percentage of responding patients, time to recurrence or overall survival for patients receiving chemotherapy with ATRA vs. historical controls receiving chemotherapy without ATRA. After in vitro exposure of day 3 bone marrow samples to ATRA, there was an increase in apoptotic cells in 25% of patient samples compared with samples not exposed to ATRA. Later date of peak apoptosis in peripheral blood and higher percentage of apoptotic cells in bone marrow on day 3 of treatment were associated with lack of clinical response to treatment. Increased bcl-2 in patient samples was associated with shorter time to recurrence and poor cytogenetic risk. The addition of ATRA to chemotherapy did not improve patient outcome. However, evidence of in vitro response to ATRA in 25% of patients suggests that retinoid pathways should be studied further in patients with AML.

Differences in induction of p53, p21WAF1 and apoptosis in relation to cell cycle phase of MCF-7 cells treated with camptothecin.

The DNA topoisomerase I (topI) inhibitor camptothecin (CPT), stabilizes so-called cleavable complexes which consist of topI covalently attached to 3' OH ends of DNA nicks. Collisions between the progressing DNA replication forks (occurring in S phase cells) or between the transcription driven RNA polymerase molecules (occurring in G1, S and G2 cells) and these complexes convert the latter into secondary DNA lesions which are unrepairable and lethal to the cell. Changes induced by CPT in the level of the tumor suppressor p53, cyclin-dependent kinase inhibitor p21WAF1 and proapoptotic protein Bax (all detected immunocytochemically), were measured separately in the nucleus and cytoplasm of individual human breast carcinoma MCF-7 cells by laser scanning cytometry (LSC) in relation to cell cycle position and induction of apoptosis. The initial transient cell arrest at the G1 checkpoint seen at 8-16 h of treatment with 0.15 microM CPT was accompanied by the rapid accumulation of p53 (preventable by cycloheximide) in the nucleus; the rise (>20-fold) in p53 was maximal for S phase cells. The magnitude of the nuclear p53 increase induced by CPT, at maximum, was 2-fold higher than that induced by the proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (LLnL). While the accumulation of p53 was seen in all phases of the cycle, only G1 cells responded by induction ( approximately 60-fold increase) of p21WAF1. Inhibition of DNA replication by aphidicolin prevented the accumulation of p53 in S and G2/M but had no effect on its induction in G1 cells. Perturbation of cell progression through S phase was seen between 24-72 h of treatment, and it coincided with induction of Bax and apoptosis (both maximal in S phase cells). Thus, the changes observed in S phase cells (nuclear accumulation of p53 preventable by aphidicolin, induction of Bax, apoptosis), triggered by the collisions of DNA replication forks with the CPT-induced lesions, were distinct from the changes in G1 (nuclear p53 accumulation unaffected by aphidicolin, induction of p21WAF1) presumably triggered by collisions of RNA polymerase with the CPT-lesions. Great heterogeneity in expression of p53 and p21WAF1 of the G1 cell population in response to CPT was observed, which may reflect the intercellular variability in the rate of transcription (i.e., frequencies of collisions of RNA polymerase with the lesions). Thus, differences in the transcriptional activity of G1 cells may play a role in their sensitivity to CPT and similar topI inhibitors.

High affinity binding of fluorescein isothiocyanate to eosinophils detected by laser scanning cytometry: a potential source of error in analysis of blood samples utilizing fluorescein-conjugated reagents in flow cytometry.

BACKGROUND: In samples of peripheral blood cells processed using the commercial kits for detection of apoptosis based on DNA strand break labeling, a subpopulation of cells characterized by high green fluorescence, similar in intensity to that of apoptotic cells but more uniform, was consistently observed by flow cytometry. The labeled cells had no other features of apoptosis. The labeling was observed regardless of the fixative used and was evident in control samples lacking terminal deoxynucleotidyltransferase. Common to all the kits that generated this labeling pattern was the presence of fluorescein (f) conjugated reagents, f-dUTP, f-avidin, or f-antibody. METHODS: Laser scanning cytometry was used to identify the labeled cells and study the mechanism of labeling. Because it was suspected that the traces of unconjugated f-isothiocyanate (FITC) that may contaminate the reagents were responsible for the labeling, FITC binding affinity to white blood cells was studied. Gel electrophoresis was used to detect the presence of unconjugated FITC in the reagents. RESULTS: After staining with Giemsa, the strongly fluorescent objects were identified as eosinophils with normal morphology and no evidence of apoptosis. The fluorescence was localized exclusively within the cytoplasmic granules. Labeling of eosinophils was observed at 2 nM concentration of FITC, which was over three orders of magnitude lower than that needed to label neutrophils, monocytes, or lymphocytes. Gel electrophoresis of the f-conjugated reagents revealed only minor contamination with FITC. CONCLUSIONS: (1) Trace amounts of unconjugated FITC contaminating the reagents are adequate to strongly label eosinophils thereby introducing experimental bias in analysis of apoptosis and in other studies on blood cells utilizing f-labeled antibodies, e.g., in detecting cytokines. (2) FITC at concentration 2-500 nM can be used as a marker of eosinophiles; (3) Because of high affinity to FITC, eosinophiles (or the protein from these cells) may serve as a means of removing traces of unconjugated FITC from the reagents during their manufacture or prior to use.

Malignancy: A New Approach to the Analysis of Apoptosis in the Leukemic Subpopulation by Flow Cytometry Using a CD45 Gating Strategy.

Bone marrow and peripheral blood are heterogeneous tissues containing cells of different hematopoietic lineages. It is possible to detect leukemic cells by flow cytometry using a gating strategy, which combines CD45 expression on the cell surface with right angle light scatter (SS). This approach was applied to 15 cases of AML. Myeloblasts had the lowest CD45 fluorescence intensity of any of the cells in the myeloid series and also had the lowest SS, approximately equivalent to monocytes, but greater than lymphoblasts and lymphocytes. Using this gating strategy in each sample we could identify up to 5 separate cell compartments. Our results showed good correlation between the flow differential and the manual differential cell count. However in some cases, especially when a sample became hypocellular, the flow differential was more sensitive in identifying leukemic blasts. Total apoptosis (i.e. apoptosis in all cell populations combined) varied during the treatment between 0-34%. In the blood, the highest percentage of total apoptotic cells usually occurred between day 3-5 of treatment. The percentage of apoptotic cells varied depending on the cell type on a percentage basis. The leukemic population was lesslikely to undergo apoptosis compared to the lymphocytes, monocytes and more mature myeloid cells. In normal cells, apoptosis occurred mostly in G(1) and S phases of the cell cycle. Apoptosis among CD45-blasts usually varied between 0-5%. Myeloblasts also had a tendency to undergo apoptosis in G(1) and S phases of the cell cycle. The CD45-blast apoptotic peak in the blood occurred between day 5-7 of treatment. Analysis of drug-induced apoptosis in bone marrow seems to provide more information than such measurements in peripheral blood.

Activation of nuclear factor kappa B (NF-kappaB) assayed by laser scanning cytometry (LSC).

Nuclear factor kappa B (NF-kappaB)/rel is the family of ubiquitous transcriptional activators involved in regulation of diverse immune and inflammatory responses. It also plays a role in control of cell growth and apoptosis. In its inactive form NF-kappaB remains in the cytoplasm sequestered through interaction with IkappaB protein. Rapid translocation of NF-kappaB from cytoplasm to nucleus that occurs in response to extracellular signals is considered to be a hallmark feature of its activation. The translocation of NF-kappaB in HL-60, U-937 and Jurkat leukemic cells as well as in human fibroblasts induced by tumor necrosis factor alpha (TNF-alpha) or phorbol myristate acetate (PMA) was presently measured by laser scanning cytometry (LSC). NF-kappaB was detected immunocytochemically with FITC-tagged antibody and its presence in the nucleus vis-a-vis cytoplasm was monitored by measuring the green fluorescence integrated over the nucleus, which was counterstained with propidium iodide (PI), and over the cytoplasm, respectively. Activation of NF-kappaB led to a rapid increase in NF-kappaB-associated fluorescence measured over the nucleus (FN) concomitant with a decrease in fluorescence over the cytoplasm (F(C)), which was reflected by an increase in F(N)/F(C) ratio. This rapid assay of NF-kappaB activation can be combined with morphological identification of the activated cells or with their immunophenotype. Bivariate analysis of NF-kappaB expression versus cellular DNA content makes it possible to correlate its activation with the cell cycle position. The described method has a potential to be used as a functional assay to monitor intracellular translocation of other transcriptional activators such as p53 tumor suppressor protein or signal transduction molecules.

Enhanced in vitro cytotoxicity and cytostasis of the combination of onconase with a proteasome inhibitor.

In proliferating cells the turnover rate of proteins responsible for regulation of the cell cycle progression, namely cyclins and inhibitors of the cyclin-dependent kinases (CDKs) and phosphatases, is rapid and their cellular level is modulated at the transcriptional, translational and/or degradation (via proteasome pathway) stages. Inhibition of proteasome function results in accumulation of rapidly turning over proteins and, thus, causes an imbalance of the cell cycle regulatory components, and loss of their regulatory function. Indeed, it has been shown that proteasome inhibitors perturb the cell cycle progression. Onconase, a novel RNase which has anti-tumor activity and is in clinical trials, has previously been shown to suppress protein synthesis, presumably by degradation of intracellular RNA, preferentially tRNA. By interfering with regulation of expression of cyclins and/or CDK-inhibitors, onconase also may induce the imbalance of these proteins and potentiate the effect of proteasome inhibitors. In the present study, we observed that the combinations of onconase with peptide-aldehyde inhibitors of calpain and proteasome such as the N-acetyl-leucinyl-leucinyl-norleucinal (LLnL) and the N-acetyl-leucinyl-valinyl-phenylalaninal (LVP), but not N-acetyl-leucinyl-leucinyl-methioninal (LLM), were synergistic in suppressing cell proliferation and inducing apoptosis in three human tumor cell lines: A-549 lung adenocarcinoma, DU-145 prostatic carcinoma, and MDA-MB-231 breast carcinoma. The observed cytotoxicity may also be a result of prevention of the induction of the 'survival' genes by the nuclear factor kappaB (NFkappaB) by onconase and proteasome inhibitors. The data indicate that such combinations should be further tested as potential anti-cancer regimens.

Potentiation of tumor necrosis factor induced apoptosis by onconase.

Onconase (ONC) a ribonuclease from amphibian oocytes is cytostatic and cytotoxic to many human tumor lines, shows in vivo antitumor activity in mouse tumor models and is in Phase III clinical trials. The mechanism of antitumor activity of ONC is presumed to be due to its internalization, degradation of intracellular RNA and suppression of protein synthesis. Since apoptosis triggered by TNF-alpha is known to be potentiated by inhibitors of protein synthesis, we have hypothesized that it also may be potentiated by ONC. Indeed, preincubation of U-937 or HL-60 leukemic cells with 0.17 microM ONC rendered them more sensitive to induction of apoptosis by TNF-alpha or antibody to CD95 (Fas). The mechanism by which ONC amplifies the effect of TNF-alpha may involve suppression of induction of the survival genes whose expression is triggered by activation of NFkB by this factor.