Overexpression of bax sensitizes breast cancer MCF-7 cells to cisplatin and etoposide

Bax, one of the bcl-2 family genes, is expressed in a number of untransformed cell lines and various breast tissues, whereas only weak or no expression has been detected in breast cancer cell lines and malignant breast tissue. Human breast cancer MCF-7 cells, which have a weak bax gene expression, were stably transfected with pCX2neo bax, encoding human bax; and two unique clones, MCF-7/bax-1 and MCF-7/ bax-2, that expressed different levels of bax were generated. Sensitivity to cisplatin (CDDP) and etoposide (VP-16) was examined and each stable transfectant was more sensitive to these agents than the parental MCF-7 cells. The degree of enhancement in sensitivity to these anticancer agents was dependent on the expression level of bax. The enzyme-linked immunosorbent assay (ELISA), which quantifies DNA damage, demonstrated that this sensitization was due to apoptosis. Thus, we suggest that exogenous bax-alpha overexpression may be one of the factors determining cellular chemosensitivity in MCF-7 breast cancer cells and that it could be applied therapeutically to enhance chemosensitivity in breast cancer cells.     

Impairment of Fas-antigen expression in adriamycin-resistant but not TNF-resistant MCF7 tumor cells

The proteinase inhibitor set in skeletal muscle is poorly characterized at present. This study was aimed to investigate in mouse skeletal muscle 1) the tissue-associated counterpart, if any, of serum protease inhibitors (which may also play antiproteolytic functions in tissues) and 2) calpastatin, a tissue inhibitor of calcium-activated neutral proteases (calpains). Triton-extracts were prepared from muscle homogenates of mice, which had been perfused extensively with phosphate buffered saline (PBS) (under deep anesthesia) to remove blood inhibitors. Among various inhibitors tested, the following muscle-associated inhibitors were identified by western-blotting: alpha-2-macroglobulin (185, 165, 35 kDa), alpha-1-antitrypsin (52 kDa), inter-alpha-trypsin inhibitor (220, 180 kDa) and calpastatin (70 kDa). Combined light microscope and confocal immunohistochemical experiments revealed that, in all muscles examined (soleus, plantaris, extensor digitorum longus) the above specific immunoreactivities were localized outside the muscle fibers (in periendomysium, blood vessel wall) as well as within them. Inter-alpha-trypsin inhibitor, however, completely lacked the intracellular localization. This wide distribution of proteinase inhibitors suggests that numerous muscular structures may be normally protected from unwanted proteolysis, thus providing an essential background for further studies on pathological models with altered proteolysis (m. dystrophy, denervation atrophy, etc.).     

Autoimmune haemolytic anaemia responding to anti-tuberculous treatment

We describe a very uncommon case of gastric duplication with heterotopic pancreas suggested by an acute pancreatitis and complicated by pseudocyst formation. Diagnostics, hypothesis and management are discussed.     

NGF, cyclic AMP, and phorbol esters regulate oxytocin receptor gene transcription in SK-N-SH and MCF7 cells

In an initial study, the effects of galactose intoxication on nerve laser Doppler blood flow (NLDF) and nerve conduction velocity (NCV) were assessed after 1-16 weeks of galactose feeding in pentobarbital-anesthetized rats. NLDF was not significantly changed at any time point. NCV was significantly reduced after 16, but not 1 or 4, weeks of galactose feeding. In a second study, NLDF was not significantly changed by 4 weeks of galactose intoxication, but streptozotocin-diabetic NLDF was significantly reduced compared to both control (P<0.001) and galactose-intoxicated rats (P<0.05). Compared to control animals, sciatic motor NCV was significantly (P<0.001) reduced in the galactose group, while sciatic and saphenous sensory NCVs were not significantly changed. In the streptozotocin-diabetic rats, motor and sensory NCVs were all significantly reduced (P<0.001). In contrast to the NCV findings, mean caliber of myelinated axons in both the saphenous and sciatic nerves was reduced in galactose-intoxicated, but not streptozotocin-diabetic rats. The observed sequence of changes associated with these two models of diabetic neuropathy is not consistent with the proposed roles of ischemia and axonal dwindling in the reported nerve conduction deficits.     

Interactions between biospecific polymers and MCF7 cells: modulation of cellular proliferation and expression of estrogen receptors

Numerous studies on interactions between insoluble polymers and cell membrane receptors indicated modulation of cellular proliferation and cell phenotype by these polymers considered as biospecific. We synthesized several biospecific polymers in order to investigate the interactions between polymers and intracellular receptors as estrogen receptors considered as tumoral indicator of breast cancer. Biospecific polymers were obtained by random substitutions of crosslinked polystyrene beads with suitable chemical groups (sulfonate and amino acid sulfamides). These polymers were used as microcarriers for culture of MCF7 cells, a cellular model of human breast cancer. Quantification of MCF7 cell estrogen receptors was determined by radioligand binding assay for different days of cellular proliferation. The data obtained with MCF7 cells cultured on biospecific polymers show an inverse relationship between polymer induced inhibition of cell proliferation and polymer induced increase of estrogen receptors. Similar inverse relationship was obtained with MCF7 cell cultured on standard polystyrene tissue culture plates. The various interaction between insoluble polymers and MCF7 cells could be related to the proportion and the nature of the substitutive chemical groups. These biospecific polymers could presents sites of interaction with cell membrane receptors leading to modulation of cell biological activity. The different insoluble polymers were used as preliminary models: a practical application could be a methodology of cellular selection using soluble biospecific polymers (for example chemically modified dextrans).     

Enhancement of tamoxifen-induced E-cadherin function by Ca2+ channel antagonists in human breast cancer MCF7/6 cells

Despite its intensive use in adjuvant breast cancer therapy for more than 30 years, the exact mechanisms of action of tamoxifen have not yet been fully characterized. Tamoxifen was recently shown to restore the E-cadherin function of human breast cancer MCF7/6 cells and to suppress their invasive phenotype. Because tamoxifen interacts with targets implicated in Ca2+ homeostasis, we explored the possibility that the restoration of E-cadherin function in MCF7/6 cells induced by this drug could be affected by Ca2+ modulators. Two different Ca2+ channel antagonists (verapamil and nifedipine) potentiated the effect of tamoxifen on E-cadherin function, as evaluated with a fast cell aggregation assay. These molecules decreased the tamoxifen concentration needed to restore the E-cadherin function from 10(-6) M to 10(-7) M. When incubated with a Ca2+ channel agonist, Bay K8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoro-methylphenyl)- pyridine-5-carboxylate), the effect of tamoxifen on E-cadherin function was completely abolished. These results demonstrate that the restoration of the E-cadherin function induced by tamoxifen depends, at least in part, on a Ca2+ pathway, and support the evidence of an effect of tamoxifen on Ca(2+)-dependent mechanisms. Our data also suggest that Ca2+ channel modulators could make it possible to decrease the dose of tamoxifen administered to patients without reducing the therapeutic effects.     

Alteration of the sphingomyelin/ceramide pathway is associated with resistance of human breast carcinoma MCF7 cells to tumor necrosis factor-alpha-mediated cytotoxicity

The interference of tumor necrosis factor-alpha (TNF) signaling processes with the acquisition of tumor resistance to TNF was investigated using the TNF-sensitive human breast carcinoma MCF7 cell line and its established TNF-resistant variant (R-A1). The resistance of R-A1 cells to TNF correlated with a low level of p55 TNF receptor expression and an absence of TNF signaling through TNF receptors. Stable transfection of wild-type p55 receptor in R-A1 resulted in enhancement of p55 expression and in partial restoration of TNF signaling, including nuclear factor-kappaB (NF-kappaB) activation. However, the transfected cells remained resistant to TNF-induced apoptosis. Northern blot analysis revealed a comparable induction of manganous superoxide dismutase and A20 mRNA expression in p55-transfected cells and in sensitive MCF7 cells, making it unlikely that these genes are involved in the resistance to TNF-mediated cytotoxicity. While TNF significantly stimulated both neutral and acidic sphingomyelinase (SMase) activities with concomitant sphingomyelin (SM) hydrolysis and ceramide generation in MCF7, it failed to trigger these events in TNF-resistant p55-transfected cells. In addition, the basal SM content was significantly higher in sensitive MCF7 as compared to the resistant counterparts. Furthermore, the TNF-resistant cells tested could be induced to undergo cell death after exposure to exogenous SMase or cell-permeable C6-ceramide. This study also shows that TNF failed to induce arachidonic acid release in p55-transfected resistant cells, suggesting that an alteration of phospholipase A2 activation may be associated with MCF7 cell resistance to TNF. Our findings strongly suggest a role of ceramide in the mechanism of cell resistance to TNF-mediated cell death and may be relevant in elucidating the biochemical nature of intracellular messengers leading to such resistance.     

Bcl-xL functions downstream of caspase-8 to inhibit Fas- and tumor necrosis factor receptor 1-induced apoptosis of MCF7 breast carcinoma cells

Stimulation of the Fas or tumor necrosis factor receptor 1 (TNFR1) cell surface receptors leads to the activation of the death effector protease, caspase-8, and subsequent apoptosis. In some cells, Bcl-xL overexpression can inhibit anti-Fas- and tumor necrosis factor (TNF)-alpha-induced apoptosis. To address the effect of Bcl-xL on caspase-8 processing, Fas- and TNFR1-mediated apoptosis were studied in the MCF7 breast carcinoma cell line stably transfected with human Fas cDNA (MCF7/F) or double transfected with Fas and human Bcl-xL cDNAs (MCF7/FB). Bcl-xL strongly inhibited apoptosis induced by either anti-Fas or TNF-alpha. In addition, Bcl-xL prevented the change in cytochrome c immunolocalization induced by anti-Fas or TNF-alpha treatment. Using antibodies that recognize the p20 and p10 subunits of active caspase-8, proteolytic processing of caspase-8 was detected in MCF7/F cells following anti-Fas or TNF-alpha, but not during UV-induced apoptosis. In MCF7/FB cells, caspase-8 was processed normally while processing of the downstream caspase-7 was markedly attenuated. Moreover, apoptosis induced by direct microinjection of recombinant, active caspase-8 was completely inhibited by Bcl-xL. These data demonstrate that Bcl-xL can exert an anti-apoptotic function in cells in which caspase-8 is activated. Thus, at least in some cells, caspase-8 signaling in response to Fas or TNFR1 stimulation is regulated by a Bcl-xL-inhibitable step.     

Channeling of three newly introduced antidepressants to patients not responding satisfactorily to previous treatment

OBJECTIVE: Trifluoromethane and CO are produced simultaneously during the breakdown of isoflurane and desflurane by dry CO2 absorbents. Trifluoromethane interferes with anesthetic agent monitoring, and the interference can be used as a marker to indicate anesthetic breakdown with CO production. This study tests representative types of gas monitors to determine their ability to provide a clinically useful warning of CO production in circle breathing systems. METHODS: Isoflurane and desflurane were reacted with dry Baralyme at 45 degrees C. Standardized samples of breakdown products were created from mixtures of reacted and unreacted gases to simulate the partial degrees of reaction which might result during clinical episodes of anesthetic breakdown using 1% or 2% isoflurane and 6% or 12% desflurane. These mixtures were measured by the monitors tested, and the indication of the wrong agent or a mixture of agents due to the presence of trifluoromethane was recorded and related to the CO concentration in the gas mixtures. RESULTS: When presented with trifluoromethane from anesthetic breakdown, monochromatic infrared monitors displayed inappropriately large amounts of isoflurane or desflurane. Agent identifying infrared and Raman scattering monitors varied in their sensitivity to trifluoromethane. Mass spectrometers measuring enflurane at mass to charge = 69 were most sensitive to trifluoromethane. CONCLUSION: Monochromatic infrared monitors were unable to indicate anesthetic breakdown via interference by trifluoromethane, but did indicate falsely elevated anesthetic concentrations. Agent identifying infrared and Raman monitors provided warning of desflurane breakdown via the interference of trifluoromethane by displaying the wrong agent or mixed agents, but may not be sensitive enough to warn of isoflurane breakdown Some mass spectrometers provided the most sensitive warnings to anesthetic breakdown via trifluoromethane, but additional data processing by some patients monitor units reduced their overall effectiveness.     

Altered responding to cholecystokinins and dopaminergic agonists following 6-hydroxydopamine treatment in rats.

In 5 experiments with 265 male Wistar albino rats, production of lesions in the brain dopamine (DA) system by intraventricular injection of 6-hydroxydopamine (6-OHDA) resulted in increased responses to subcutaneous apomorphine (0.5 mg/kg) and reduced responses to methamphetamine (0.15 mg/kg). It also made Ss increase responding to intracerebroventricular (icv) cholecystokinin octapeptide (CCK-8; 0.5–2 μg) and reduce responding to cholecystokinin tetrapeptide (CCK-4; 0.5–2 μg). Response changes were quantified by measuring the level of general activity. Results indicate that DA dysfunction not only affected DA receptor sensitivity but also the sensitivity of the CCK system. The response to CCK-8 was partially blocked by a selective CCK-8 antagonist, proglumide (5 μg, icv), a result suggesting the involvement of the CCK-8 receptor system. Results indicate that manipulation of 1 neuronal system could induce sensitivity changes in another closely related system. (32 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Caspase activation is required for nitric oxide-mediated, CD95(APO-1/Fas)-dependent and independent apoptosis in human neoplastic lymphoid cells

Nitric oxide (NO), an important effector molecule involved in immune regulation and host defense, was shown to induce apoptosis in lymphoma cells. In the present report the NO donor glycerol trinitrate was found to induce apoptosis in Jurkat cells that are sensitive to CD95-mediated kill. In contrast, a CD95-resistant Jurkat subclone showed substantial protection from apoptosis after exposure to NO. NO induced mRNA expression of CD95 (APO-1/Fas) and TRAIL/APO-2 ligands. Moreover, NO triggered apoptosis in freshly isolated human leukemic lymphocytes which were also sensitive to anti-CD95 treatment. The ability of NO to induce apoptosis was completely blocked by a broad-spectrum ICE (interleukin-1beta converting enzyme)-protease/caspase inhibitor and correlated with FLICE/caspase-8 activation. This activation was abrogated in some neoplastic lymphoid cells but not in others by the inhibitor of protein synthesis cycloheximide. Our results were confirmed using an in vitro experimental model of coculture of human lymphoid target cells with activated bovine endothelial cells generating NO as effectors. Furthermore, the inhibition of endogenous NO production with the inducible NO synthase inhibitor NG-monomethyl-L-arginine caused a complete abrogation of the apoptotic effect. Our data provide evidence that NO-induced apoptosis in human neoplastic lymphoid cells strictly requires activation of caspases, in particular FLICE, the most CD95 receptor-proximal caspase. Depending on the cell line tested this activation required or was independent of the CD95 receptor/ligand system.     

Estrogen treatment induces elevated expression of HMG1 in MCF-7 cells

Cytokines are thought to mediate the catabolic states induced by infection and trauma. Recent evidence suggests that the cytokine interleukin-1 beta (IL-1 beta) directly inhibits the anabolic insulin-like growth factor (IGF)-I: growth hormone (GH) axis. The biological activity of circulating IGF is regulated by the hepatocyte derived, GH-dependent acid-labile subunit (ALS) of the 140-kDa IGF binding protein (IGFBP) complex. ALS buffers the growth and metabolic effects of the insulin-like growth factors by sequestering them in a ternary complex with IGFBP-3. To determine whether IL-1 beta has a direct effect on hepatic ALS production, we have examined its effect on ALS mRNA levels and secretion in hepatocytes under GH-induced and basal conditions. In the presence of GH (30 ng/mL) half-maximal reduction of ALS mRNA levels and secretion was induced by between 0.3-3 ng/mL rhIL-1 beta (P < 0.05). However, under basal conditions IL-1 beta had no significant effect on ALS mRNA levels, and only a slight suppression of secretion. Our study suggests that IL-1 beta regulates ALS gene expression and secretion in a way that is dependent, in part, on interaction with the GH signalling pathway.     

Supplementation of MCF-7 cells with essential fatty acids induces the activation of protein kinase C in response to IGF-1

V(D)J recombination consists of a DNA cleavage reaction catalysed by RAG1 and RAG2, followed by an end-joining reaction that utilizes the cell's double-strand break repair machinery. Genes essential for the end-joining reaction include: XRCC4 encoding a protein of unknown enzymatic function; XRCC5 and XRCC6 encoding 86 and 70 kDa subunits of the Ku autoantigen, a DNA end-binding protein that is also the regulatory subunit of DNA-dependent protein kinase (DNA-PK); and XRCC7 encoding the catalytic subunit (DNA-PKcs) of DNA-PK. Recent progress in understanding the cleavage reaction, coupled with what was previously known about Ku, DNA-PK, and double-strand break repair, provide the foundation for a working model of how V(D)J recombination might be catalysed.     

Response of MCF7 human breast cancer to tamoxifen: evaluation by the three-time-point, contrast-enhanced magnetic resonance imaging method

Variations in the cellular volume fraction and in the microvascular permeability of MCF7 human breast tumors were used to assess response to tamoxifen. These pathophysiological features were mapped by applying the three-time-point, contrast-enhanced, high resolution magnetic resonance imaging method (H. Degani et al, Nat. Med., 3: 780-782, 1997). Short-term treatment with tamoxifen caused a highly significant increase in the fraction of pixels displaying intermediate contrast agent clearance pattern and a significant increase in the fraction of pixels displaying high rate of contrast agent entrance. These changes resulted from a marked rise in the extracellular volume fraction, indicating increased necrosis, and from an augmentation in the microvascular permeability, predominantly in the vicinity of the high extracellular volume fraction areas, as a result of stress-induced angiogenesis.     

Chronic oral etoposide and tamoxifen in the treatment of far-advanced hepatocellular carcinoma

BACKGROUND: Hepatocellular carcinoma (HCC) is a chemoresistant tumor that frequently expresses a high level of p 170 glycoprotein of the multidrug-resistance (MDR) gene. Preliminary data suggested that VP-16 showed modest activity in HCC. Recently, schedule-dependent cytotoxicity of VP-16 has been demonstrated. In this study, we tested the therapeutic efficacy of chronic oral VP-16 plus tamoxifen, a potential MDR-reversing agent, in patients with far-advanced HCC. METHODS: A prospective single-arm study was conducted in the National Taiwan University Hospital. To be eligible, patients must have had unresectable and non-embolizable HCC, objectively measurable tumors, adequate hemogram with absolute granulocyte count greater than or equal to 2,000/mm3, and platelet count greater than or equal to 1x10 (5)/mm3, total serum bilirubin less than or equal to 3.0 mg/dl, age less than or equal to 75 years, and a Karnofsky performance status of greater then or equal to 50%. The treatment included VP-16 (Bristol-Myers-Squibb, Princeton, NJ), 50 mg/m2/day, orally, Days 1 to 21, and tamoxifen (Pharmachemie B.V. Haarlem, Netherlands), 40 mg/day, orally, Days 1 to 21; repeated every 5 weeks. RESULTS: Between December 1990 and December 1993, a total of 33 patients were enrolled in the study. There were 28 men and 5 women, with a median age of 51 years. They received an average of 3.2 (range: 1-10) courses of chemotherapy. ECOG (Eastern Cooperative Oncology Group) Grade 3 and Grade 4 leucopenia developed in 6 patients (18.2%) and 4 (12.1%) patients, respectively. Grade 3 and 4 thrombocytopenia developed in 2 patients (6.1%). Treatment-related death occurred in one patient due to sepsis. Mild gastrointestinal toxicities were common with Grade 1 and 2 nausea. Grade 1 and 2 vomiting, Grade 1 and 2 diarrhea, and Grade 1 and 2 stomatitis, developed in 13 (39.4%), 7 (21.2%), 12 (36.4%), and 16 (48.5%) patients, respectively. Grade 3 and 4 gastrointestinal toxicities were rare. Deep vein thrombosis occurred in one patient (3.0%). Eight patients (24.2%, 95% confidence interval 11%-42%) had achieved a partial remission, with a median time-to-progression of 6 months (2-11). Median survivals of the responders and non-responders were 8.0 and 3.0 months, respectively (P < 0.05). The median Karnofsky performance status of the responders improved from 70% to 80%. CONCLUSIONS: Chronic oral VP-16 and tamoxifen has modest activity and acceptable toxicity in far-advanced HCC, and is a useful palliative treatment in about a quarter of such patients.     

Synergistic interactions of etoposide and interleukin-1 alpha are not due to DNA damage in human melanoma cells

Family nursing, always a component of nursing, has been recently receiving increased attention through publications, educational programs, clinical practice settings and family nursing research. Nurses are in the best position to assist families experiencing the illness of a loved one, but often lack the skills and confidence necessary to assist with the psychosocial needs. The purpose of this paper is to describe the development and implementation of a family nursing program within a large, tertiary care facility. The program's evaluation is ongoing but early outcomes indicate that day-to-day nursing practice has been positively influenced and improved job satisfaction may result.