Heat shock provides delayed protection against oxidative injury in cultured human umbilical vein endothelial cells

During both mild and severe ischemia, vascular endothelial cells lining large and small vessels of the ischemic organ are exposed to oxygen-derived free radicals resulting in oxidative damage to the organ. Heat shock has been shown to induce thermotolerance and also protect against ischemic injury, possibly via increased synthesis of heat shock proteins (HSPs). We hypothesized that heat shock preconditioning may protect human endothelial cells against oxidative damage. Cultured human umbilical vein endothelial cells (HUVEC) were subjected to heat shock (42 degrees C, 1 h) and allowed to recover for 2 or 20 h, at which times the cells were oxidatively stressed for 1 h by exposing them to 100-200 mumol/l of hydrogen peroxide (H2O2). Cellular damage was assessed immediately and 18 h later by morphology and release of lactate dehydrogenase (LDH). No protection of HUVEC was seen using the 2-hour recovery interval, but a significant protection (P < 0.05) was observed after the 20-hour delay. Northern blot analysis at 1 and 2 h after heating showed induction of HSP-70 mRNA. Western blot analysis demonstrated a significant increase in HSP-72 protein after 2 as well as 20 h of recovery from heat shock, although the amounts of protein at the two times were not significantly different. Furthermore, no differences in the activity of the antioxidant enzyme catalase were observed between heated and unheated HUVEC at 2 and 20 h after heat preconditioning. Thus, heat shock preconditioning induces delayed protection against oxidative injury in HUVEC, and the mechanism of protection appears to involve more than the expression of HSP-72 or activity of catalase.     

Protective effects of FK506 against glutamate-induced neurotoxicity in retinal cell culture

Nanomoles of neuropeptide Y (NPY) and noradrenaline (NA), administered i.v. to pentobarbital-anesthetized rats, caused nearly equipotent dose-dependent pressor responses in normotensive rats. However, in renovascular Goldblatt hypertensive rats, the dose-response curves for both NPY and NA were significantly displaced to the left, approximately threefold. Intravenous administration of BIBP 3226 (30-180 microg/kg) did not consistently lower blood pressure, per se, but did evoke competitive antagonism of the NPY pressor response in both rat populations. The magnitude of the NPY antagonism evoked by BIBP 3226 was comparable in normotensive and hypertensive rats. The absence of NA antagonism demonstrates the selectivity of the BIBP 3226 blockade.     

Mechanisms of protection from menadione toxicity by 5,10-dihydroindeno[1,2,-b]indole in a sensitive and resistant mouse hepatocyte line

Established cell lines derived from newborn livers of c14CoS/c14CoS and cch/cch mice have been shown to be genetically resistant (14CoS/14CoS cells) or susceptible (ch/ch cells) to menadione toxicity. These differences are due in part to relatively higher levels of reduced glutathione (GSH) and NAD(P)H:menadione oxidoreductase (NMO1) activity in the 14CoS/14CoS cells. The indolic membrane-stabilizing antioxidant 5,10-dihydroindeno[1,2-b]indole (DHII) was shown previously to protect against various hepatotoxicants in vivo and in primary rat hepatocytes. This report describes how the 14CoS/14CoS and ch/ch cell lines provide a valuable experimental system to distinguish the mechanism of chemoprotection by DHII from menadione toxicity. The addition of 25 microM DHII produced a time-dependent decrease in menadione-mediated cell death in 14CoS/14CoS cells, with little effect on ch/ch cell viability. The maximum protective effect occurred at 24 hr, although the concentration of DHII remained constant for 48 hr. The protective effect of DHII correlated with enhanced glutathione levels (234% increase at 24hr), as well as induction of four enzymes involved in the detoxification and excretion of menadione: NAD(P)H:menadione oxidoreductase (NMO1, quinone reductase), glutathione reductase, glutathione transferase (GST1A1), and UDP glucuronosyltransferase (UGT1*06), with 24-hr maximum induction of 707, 201, 171 and 198%, respectively. Other biotransformation enzymes not directly involved in menadione metabolism (glutathione peroxidase, cytochromes P4501A1 and P4501A2, copper-, zinc-dependent superoxide dismutase, and NADPH cytochrome c oxidoreductase) were not induced by DHII. Menadione-stimulated superoxide production was inhibited 50% by DHII only in 14CoS/14CoS cells, and the inhibition required 24-hr preincubation. Pretreatment with DHII also protected both cell types against the menadione-mediated depletion of GSH, and the increase in percent (oxidized glutathione GSSG), an indicator of oxidative stress. These results suggest that DHII does not protect against menadione toxicity by virtue of its antioxidant or membrane-stabilizing properties. Rather, it acts by inducing a protective enzyme profile that migates redox cycling and facilitates excretion of menadione.     

Tumor necrosis factor alpha regulates proliferation in a mouse intestinal cell line

BACKGROUND & AIMS: Tumor necrosis factor (TNF)-alpha is a prominent cytokine in the pathogenesis of inflammatory bowel disease, yet its effects on the intestinal epithelium remain poorly understood. This study was designed to investigate the action of TNF-alpha on intestinal cell proliferation. METHODS: Young adult mouse colon cells were studied under nontransformed conditions with epidermal growth factor, keratinocyte growth factor, insulin-like growth factor 1, or serum in the presence or absence of TNF-alpha and cell numbers determined. The expression and independent actions of the 55-kilodalton TNF-alpha R1 and 75-kilodalton TNF-alpha R2 receptors were studied by immunologic methods. RESULTS: TNF-alpha stimulated proliferation at 0.1 and 1 ng/mL and inhibited proliferation at 100 and 1000 ng/mL without altering cell viability. TNF-alpha inhibited the mitogenic effect of growth factors and epidermal growth factor receptor tyrosine phosphorylation. TNF-alpha R1 receptor agonist antibody inhibited proliferation, whereas a TNF-alpha R2 receptor-blocking antibody prevented the proliferative effect of low-dose TNF-alpha. CONCLUSIONS: TNF-alpha displays a novel influence on intestinal cell growth, stimulating proliferation at physiological concentrations and inhibiting proliferation at pathological concentrations. The regulation of intestinal cell mitogenesis by TNF-alpha seems to be mediated differentially by the two TNF-alpha receptors, with the TNF-alpha R1 receptor inhibiting proliferation and the TNF-alpha R2 receptor promoting proliferation.     

Hybridization of a human myeloma permanent cell line with mouse cells

A population of hybrid cells derived from the fusion of a permanent human myeloma cell line, which secretes complete IgE, and a subline of mouse L cells, did not secrete IgE as evidenced by sensitive immunosorbent tests. Also, the hybrid cells were observed not to contain intracellular IgE (epsilon or lambda chains) in amounts to be detectable by fluorescent antibody techniques. The doubling times and cell cycle parameters of the hybrid cells were found to be similar to those of the slow-growing parental human myeloma cells, in addition, the growth of the hybrid cells was characterized by a higher degree of contact inhibition than the parent mouse cells.     

Prostaglandin E receptor subtypes in mouse osteoblastic cell line

PGE2 is one of the key molecules in the osteoblast. It is the major prostanoid in the bone, and its production is under the control of both systemic and local factors. PGE2 has been reported to have multiple actions in the osteoblast, such as growth promotion and cell differentiation. To better understand the action of PGE2 in the osteoblast, we determined the PGE receptor subtypes in MC3T3-E1, an osteoblastic cell line derived from the normal mouse calvaria. Northern blot analysis revealed that EP1 and EP4 subtypes are expressed in MC3T3-E1. In contrast, EP3 subtype was not detected by either Northern blot analysis or RT-PCR. The contribution of each subtype was evaluated by studying the effects of subtype-specific analogs on osteoblastic function at confluency and 5 days after confluency. An EP1 agonist, 17-phenyl-omega-trinor PGE2, increased DNA synthesis and decreased alkaline phosphatase activity. 11-Deoxy-PGE1, and EP2 and EP4 agonist, decreased DNA synthesis and increased alkaline phosphatase activity at both stages. Butaprost, an EP2-selective agonist, showed effects similar to those of 11-deoxy-PGE1 only at confluency. Another and more differentiated osteoblastic marker, osteocalcin production, was detectable and was stimulated by 11-deoxy-PGE1 only 5 days after confluency. The exposure of these cells to EP1 agonist changed the cell shape to a more fibroblastic appearance. These results indicate that EP1, EP4, and probably EP2 are present in MC3T3-E1 cells; EP1 promotes cell growth, and EP2 and EP4 mediate differentiation of the osteoblast. Furthermore, the decreased response to EP2-specific agonist 5 days after confluency suggests that the expression of PGE receptor subtype is dependent on the stage of osteoblastic differentiation. This is the first report to determine PGE receptor subtypes in the bone.     

Lithium protection against oxygen toxicity in rats: ammonia and amino acid metabolism

1. The use of Li pre-treatment in rats before high pressure oxygen exposure has been reported effective in controlling convulsions. This is an effect which is better demonstrated if exposure to oxygen follows shortly after Li injection than exposure following several hours later. 2. This study has investigated the hypothesis that the protective action of Li may be exerted, in the short term, by its removing ammonia from the blood and alleviating the latter's known toxic action. 3. A normal Li distribution time profile in unstressed rat brain and blood following intraperitoneal injection has been established. Brain and blood ammonia, amino acids and Li concentrations were also measured in Li-treated animals exposed and convulsed by oxygen. These measurements were made both shortly (15 min) and also several hours after (24 hr) Li treatment. Ammonia and amino acid values in Li-protected groups were compared to normal unstressed animal values and also to values in animals convulsed by oxygen unprotected by Li pre-treatment. 4. In rat brain abd blood significant (P less than 0-001) elevation of ammonia and glutamine and depression of gamma-amino butyric acid (brain only) and glutamate was noted following oxygen treatment in unprotected animals. Prior injection of Li 15 min before high pressure oxygen exposure delayed convulsions twice as long. Additionally if these animals were only exposed to oxygen for a period of time equal to that which would normally produce convulsions in unprotected animals, brain and blood ammonia and amino acids were maintained near to unstressed animal levels. Concomitantly, blood Li concentrations were considerably depressed below the values one would expect from the previously determined Li distribution time profile. 5. In rats exposed to high pressure oxygen 24 hr after Li treatment there was no protective action against high pressure oxygen convulsion, rather a potentiating effect for convulsion was seen. 6. These data present compelling evidence for the controlling effect of Li in rats, on rising blood ammonia concentration which occurs in high pressure oxygen exposure. The effect might well be due to the known chelating properties of Li with ammonia.     

Antileukemia activity of a natural killer cell line against human leukemias

We describe here the in vitro and in vivo antileukemia activity of a recently described natural killer (NK) cell line (NK-92), which has features of human activated NK cells. The cytotoxic activity of rhIL2-dependent cultured NK-92 cells against primary patient-derived leukemic target cells [12 acute myelogenous leukemias (AMLs), 7 T acute lymphoblastic leukemias (T-ALLs), 14 B-lineage-ALLs, and 13 chronic myelogenous leukemias (CMLs)], human leukemic cell lines (K562, KG1, HL60, Raji, NALM6, TALL-104, CEM-S, and CEM-T) and normal bone marrow cells was measured in 51Cr-release assay (CRA). The patient-derived leukemias could be subdivided into three groups based on their sensitivity to NK-92 cells: insensitive (< or =19% lysis), sensitive (20-49% lysis), and highly sensitive (> or =50% lysis) at an E:T ratio of 9:1. Of 46 patient-derived samples, 24 (52.2%) were sensitive or highly sensitive to NK-92-mediated in vitro cytotoxicity (6 of 12 AMLs, 7 of 7 T-ALLs, 5 of 14 B-lineage-ALLs, and 6 of 13 CMLs). NK-92 cells were highly cytotoxic against all of the eight leukemic cell lines tested in a standard 4-h CRA. Normal human bone marrow hematopoietic cells derived from 18 normal donors were insensitive to NK-92-mediated cytolysis. In comparison with human lymphokine-activated killer cells, normal NK cells, and T cells, NK-92 cells displayed more powerful antileukemia activity against a patient-derived T-ALL as well as K562 and HL60 cells, both in in vitro CRA and in a xenografted human leukemia SCID mouse model. The NK-92 cells did not induce the development of leukemia in SCID mice after i.v., i.p., or s.c. inoculation. In adoptive transfer experiments, SCID mice receiving i.p. inoculations of human leukemias derived from a T-ALL (TA27) and an AML (MA26) that were highly sensitive to the cytolysis of NK-92 cells in vitro, as well as a pre-B-ALL (BA31) that was insensitive to the in vitro cytolysis of NK-92 cells, were treated by administration of NK-92 cells with or without rhIL2 (2 x 10(7) NK-92 cells i.p.; one dose or five doses). Survival times of SCID mice bearing the sensitive TA27 and MA26 leukemias were significantly prolonged by adoptive cell therapy with NK-92 cells. Some of the animals who received five doses of NK-92 cells with or without rhIL2 administration were still alive without any signs of leukemia development 6 months after leukemia inoculation. In contrast, survival of mice bearing the insensitive BA31 leukemia were not affected by this treatment. This in vitro and in vivo antileukemia effect of NK-92 cells suggests that cytotoxic NK cells of this type may have potential as effectors of leukemia control.     

Fc receptors are not required for antibody-mediated protection against lethal malaria challenge in a mouse model

The mechanisms by which Abs mediate protection during blood-stage malaria infections is controversial, with some evidence pointing to the direct effect of Abs on parasite invasion and growth, while other studies suggest that Abs act in cooperation with monocytes to achieve parasite inhibition. To determine whether the effector phase of protection in vivo to the rodent parasite Plasmodium yoelii yoelii requires Fc receptor bearing cells, we passively transferred immune sera into FcR gamma-chain knockout mice. Inflammatory macrophages from these knockout mice were unable to mediate phagocytosis or Ab-dependent cell-mediated cytotoxicity (ADCC) through Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Passive transfer of either P. y. yoelii hyperimmune sera or anti-GST-PYC2 sera directed to the major merozoite surface protein (MSP-1) of this parasite enabled both BALB/cByJ mice and FcR gamma-chain-deficient mice to resist lethal P. y. yoelii 17XL (Py17XL) challenge. mAb302, a protective IgG3 Ab, also passively protected both strains of mice. Most of these samples contain Ab isotypes that would not be able to protect mice if their protective effects required Ab-dependent cell-mediated cytotoxicity. These results establish that, in this infection, protection is directly mediated by Abs and does not require the participation of Fc receptors.     

Expression of glucocorticoid and mineralocorticoid receptors in an immortalized hippocampal neuronal cell line

A clonal cell line of rat embryonic hippocampal origin (H19-7) has been examined for the expression of glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). H19-7 cells grown at 33 degrees C continue to divide, however when grown at 39 degrees C in reduced levels of serum the cells undergo morphological differentiation and express neuronal properties. Immunocytochemistry demonstrated that H19-7 cells express both MR and GR when grown at either 33 degrees C or 39 degrees C. GR mRNA is readily detected in H19-7 cells by RNase protection assay. MR mRNA levels in H19-7 cells are too low to detect by RNase protection, but can be detected by RT-PCR. RT-PCR also demonstrated that H19-7 cells express more GR mRNA than primary hippocampal neurons. Since previous studies have shown that the level of MR mRNA is higher than that of GR mRNA in hippocampal neurons, these studies suggest that H19-7 cells represent hippocampal neurons immortalized at an early stage when the MR system is not yet fully differentiated.     

Development of a toxin-binding agent as a treatment for tunicaminyluracil toxicity: protection against tunicamycin poisoning of sheep

OBJECTIVE: To assess the ability of certain derivatives of beta-cyclodextrin to treat sheep affected by tunicaminyluracil toxicity, using tunicamycin poisoning as a model system. DESIGN: Controlled treatment trial. ANIMALS: One hundred and sixty Merino wethers were used in the studies. PROCEDURE: Groups of sheep were experimentally poisoned with tunicamycin. Derivatives of beta-cyclodextrin, with or without magnesium sulphate and magnesium gluconate, were administered to treatment groups daily for 2 to 3 days. Treatment groups were compared with untreated groups in terms of survival. RESULTS: A significant increase in survival was observed following treatment of tunicamycin-affected sheep with hydroxypropyl-beta-cyclodextrin (HP beta-CD) and magnesium sulphate in solution (P < 0.05). In subsequent trials, formulation of the cyclodextrin in the form of a magnesium gluconate gel suspension demonstrated significant protection (P < 0.01) and was equally as effective as the cyclodextrin in solution, but required half the frequency of administration, even when the treatment was not commenced until 24 h after the final toxin dose. Beta-cyclodextrin-epichlorohydrin copolymer also improved the survival rate. After toxin administration, the sheep lost significantly less weight if treatment with HP beta-CD was commenced early (P < 0.001). CONCLUSION: Protection studies using these two beta-cyclodextrin derivatives suggest that they may be effective in increasing the survival of sheep poisoned by tunicamycin and warrant further testing in field outbreaks of annual ryegrass toxicity.     

Evaluation of the efficacy of ciprofloxacin against Streptococcus pneumoniae by using a mouse protection model

A mouse protection model was used to investigate the association of the pharmacokinetics and pharmacodynamics with the in vivo efficacy of ciprofloxacin compared with that of penicillin G in the treatment of mice infected with Streptococcus pneumoniae ATCC 6303. Mice were inoculated intraperitoneally with 10 times the minimum lethal dose of S. pneumoniae. For determination of the 50% protective dose, subcutaneous antibiotics were begun 1 h after infection and were continued for 24 h. The 50% protective doses of ciprofloxacin and penicillin G were 25.52 +/- 1.95 and 0.307 +/- 0.006 mg/kg of body weight, respectively, an 83-fold difference in efficacy. For 100% protection with penicillin G, the time that the drug concentration needed to remain above the MIC was 51 min, a value easily achieved in most clinical situations. For 100% protection with ciprofloxacin, the peak concentration/MIC ratio must reach a value of 10.6. This ratio is rarely achieved with this drug against S. pneumoniae in clinical practice. These pharmacodynamic differences probably contribute to the reported differences in clinical success between these agents.     

Role of mouse and human class II transgenes in susceptibility to and protection against mouse autoimmune thyroiditis

Four cases of ocular myopathy are presented. Their clinical, electromyographical and hystological signs are discussed comparatively with clinical forms described in the literature. One case was of pure ocular myopathy, without the affectation of other skeletal muscles. The other three cases were included in the descendent ocular myopathy, presenting a motor deficiency at the centure and inferior member levels. In all cases modifyings have been found in the EMG trajectory and the muscular biopsy of skeletal and/or ocular muscles.     

Influence of heat shock on cell volume regulation: protection from hypertonic challenge in a human monocyte cell line

Heat shock response provides cells with higher tolerance against a variety of insults such as heavy metals, reperfusion injury, and endotoxin. In addition, heat treatment is known to affect ion transport mechanisms associated with vital cellular processes, including cell volume regulation. However, there has been no reports to date of a heat shock effect on cellular volume regulation itself. The aim of our study was to investigate whether the heat shock response influences volume regulation of cells. Human promonocytic U937 cells display an increase in volume in response to osmotic shrinkage. This regulatory volume increase (RVI) is mediated mainly by ion antiporters. U937 cells exposed to a temperature of 45 degrees C for 10 min (heat shock) show an enhancement of RVI after hypertonic challenge compared with untreated cells. Also, heat-treated cells display a lower intracellular pH (pHi) than untreated cells; similar control mechanisms are believed to be involved in regulating both pHi and RVI. In agreement with this, heat-shocked cells demonstrated increased activity of an HCO3(-)-independent/DIDS-sensitive pHi down-regulator, postulated to be a Cl-/HCO3- exchange. We suggest that heat shock-mediated RVI enhancement is at least partially mediated by an increased Cl-/HCO3- exchange. Our results indicate that heat shock of U937 cells activates a hitherto unknown cytoprotective effect that may help cells to overcome hypertonic challenge.     

Isolation and characterization of apoptosis-resistant mutants from a radiosensitive mouse lymphoma cell line

This paper reports the findings of a research study funded by the English National Board for Nursing, Midwifery and Health Visiting (ENB), which explored the impact of community care reforms on mental health and learning disability nurses and their practice. In this study we were struck by the divergent views of our respondents about the nature of mental health and learning disability nursing as practice disciplines and the implications of these views for the future shape of preregistration educational preparation. We noted, in particular, the debate between those who advocate what is referred to as specialist educational preparation and others who favour generic preparation. The specialist-generic debate is relevant to nursing as a whole but was magnified in the context of our study because genericism was perceived by many of our respondents as a threat to the minority branches and especially to those (arguably mental health and learning disability) that are not rooted in the biomedical tradition of general nursing. This paper seeks to contribute to this debate as it impinges on the two nursing specialties by developing models of future nurse education grounded in the empirical data from our research and interrogating them to draw out their central features. Two models are clearly independent: the 'specialist' and the 'generic' models. Another three models are partial in that they draw upon the first two: the 'pragmatic' model, the 'unity-of-nursing' model, and the 'social care' model. A common feature of the pragmatic and unity-of-nursing models is that they support the existing DipHE programme, which is now the dominant form of preregistration nurse preparation in the UK. The social care model is applicable principally to learning disability nursing.     

Benzydamine protection in a mouse model of endotoxemia

OBJECTIVE: Previous studies have shown that benzydamine (40 mg/kg s.c.) is able to inhibit tumor necrosis factor (TNF) production and to reduce mouse lethality when administered before or concomitantly with LPS. The present study was designed to further investigate benzydamine activity against LPS-induced toxicity in terms of potency and therapeutic effects. METHODS: Female Balb/c mice were used. A dose-response curve of animal lethality versus endotoxin dose was performed (LD50 = 45 micrograms/mouse). Therapeutic effects were studied selecting the dose of LPS to achieve an LD100 (160 micrograms/mouse). Mortality was assessed daily and mice were followed for 8 days. The potential mode of action of therapeutically administered benzydamine was also investigated. TNF alpha and IL-1 beta levels were measured, at 5 h after LPS injection, both in sera and in lungs. Moreover, the drug was assayed in a TNF-dependent cytoxicity test. RESULTS: Benzydamine, administered at 20 mg/kg s.c. simultaneously with the endotoxin, significantly increased LPS LD50 up to 230 micrograms/mouse (p < 0.05). Moreover, the drug significantly protected mice against LPS-induced lethality when administered either 30 min or 4 h after endotoxin injection (p < 0.001). Benzydamine, therapeutically administered at 20 mg/kg s.c., significantly reduced TNF alpha and IL-1 beta production induced by LPS both in serum and lungs and it was shown to inhibit TNF-dependent cytoxicity on L929 cells. CONCLUSIONS: These results clearly demonstrate the therapeutic activity of benzydamine in a simple model of endotoxic shock. Available data confirm the potential role of benzydamine as an anti-cytokine agent and provide suggestions for novel therapeutic applications of this anti-inflammatory drug.     

Characterization of efflux transport of organic anions in a mouse brain capillary endothelial cell line

Cumulative evidence suggests that several organic anions are actively effluxed from the brain to the blood across the blood-brain barrier (BBB). We examined the possibility of the presence of primary active transporters for organic anions (multidrug resistance associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT)) on the BBB by measuring the ATP-dependent uptake of 2,4-dinitrophenyl-S-glutathione (DNP-SG) and leukotriene C4 (LTC4) into membrane vesicles prepared from a cell line derived from mouse brain capillary endothelial cells (MBEC4). The ATP-dependent uptake of DNP-SG into the membrane vesicles was osmotically sensitive and was also supported by GTP, but not by AMP or ADP. An ATPase inhibitor, vanadate, blocked the ATP-dependent uptake of DNP-SG. The ATP-dependent uptake process was saturable, with Km values of 0.56 and 0.22 microM, and Vmax values of 5.5 and 27.5 pmol/min/mg protein for DNP-SG and LTC4, respectively. Northern and Western blot analyses showed the expression of murine MRP but not cMOAT in MBEC4 cells. Western blot analysis of the rat cerebral endothelial cells indicated the expression of protein(s) that is detectable with MRPr1, an antibody against MRP. These results, together with previous findings that both DNP-SG and LTC4 are good ligands for MRP, suggest that MRP is responsible for the unidirectional, energy-dependent efflux of organic anions from the brain into the circulating blood across the BBB.     

Isolation and characterization of a spontaneously transformed malignant mouse mammary epithelial cell line in culture

A method is described that permits the selection of spontaneously transformed mammary epithelial colonies from an untransformed mouse mammary epithelial cell line, NMuMG, and utilizes a long-term anchorage-independent growth of the transformants on soft agarose. These transformed cells (NMuMG-ST) are shown to be distinguishable from the untransformed cells by morphology, growth characteristics, induced carcinomas when transplanted into nude mice and ability to metastasize. This transformed phenotype displayed focal, multilayer growth and higher saturation density in comparison with the untransformed phenotype. Transplanted tumors as well as metastatic lung tumors in nude mice were adenocarcinomas morphologically similar to typical mammary tumors in humans. This selection procedure of mutant mammary cells from an immortalized cell line derived from normal mammary glands could be very useful to identify the genomic biomarkers in the growth regulation and malignant progression of breast cancer.