In vitro expansion of human breast cancer epithelial and mesenchymal stromal cells: optimization of a coculture model for personalized therapy approaches.

Molecularly targeted, customized therapies are designed based on the molecular portraits of cancer tissue. The efficacy of targeted therapy in individual patients depends on the contribution of single individual cancer cells within the context of their microenvironment. We have developed an in vitro model of human mammary epithelial-stromal cocultures to answer specific clinical questions related to breast cancer, to provide a tool with which to identify a signature in each breast tumor, and to identify the metabolic molecular targets of therapy in an attempt to optimize the efficacy of targeted therapy in each patient. Fifty-five human breast cancer samples were obtained through surgery. Epithelial and stromal cells were isolated from tissue specimens by differential centrifugation, and cryopreserved. Western blot analysis and RT-PCR were used to identify the tissue-specific expression patterns of cancer cells. Dose-response curves were constructed for the aromatase inhibitor formestane and for herceptin, and a 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide assay was done for combined treatment. We collected and cryopreserved, for future use, viable living cells from 55 breast tumor specimens from which we derived short-term cocultures. The presence of cytokeratins and vimentin was evaluated in 20 samples, and pHER2/neu and aromatase were evaluated in 4 cocultures. Formestane and herceptin had a cumulative growth-inhibitory effect on cocultures expressing epidermal growth factor receptors and aromatase. The in vitro model of human mammary epithelial-stromal cocultures reported herein can be used to examine, and to store, a patient's tumor-derived, living cells that retain the characteristics of the mother-tissue and respond, in vitro, to therapy.

Genes transcriptionally modulated by interferon alpha2a correlate with the cytokine activity.

BACKGROUND AND OBJECTIVES: Interferon alpha2a (IFNalpha2a) mediates important antiviral, antiproliferative and immunomodulatory responses and is employed in the treatment of human diseases, including chronic myelogenous leukemia. Here, we report the IFNalpha2a-dependent expression profiles of three malignant cell lines derived from liver, lymphocytes and muscle. DESIGN AND METHODS: The experiments were performed in the presence of cycloheximide, thus our results exclusively reflect direct transcriptional modulation. The short exposure time i.e. 5 hours evidences only the early events, excluding the effects of complex phenotypic changes on the expression. RESULTS: Our findings indicate that IFNalpha2a rapidly up-regulates the expression of STAT1, STAT2 and ISGF3G genes. This activity should result in the amplification of the cellular response to the cytokine. Moreover, IFNalpha2a directly modulates the expression of: (i) important transcriptional factors, e.g. IRF1 and IRF7 which control pivotal cellular events, and (ii) enzymes involved in the IFNalpha2a-dependent antiviral and apoptotic response. Interestingly, we showed that the cytokine induces transcriptional expression of Sjögren's syndrome antigen A1, a protein involved in several autoimmune diseases. INTERPRETATION AND CONCLUSIONS: The observed changes induced by IFNalpha2a could be related to the development of autoimmune syndromes observed during IFNalpha2a treatment. A number of genes transcriptionally regulated by the cytokine have been identified for the first time; these might represent additional effectors of IFNalpha2a activity.

Dp1 is largely dispensable for embryonic development.

E2F/DP complexes activate or repress the transcription of E2F target genes, depending on the association of a pRB family member, thereby regulating cell cycle progression. Whereas the E2F family consists of seven members, the DP family contains only two (Dp1 and Dp2), Dp1 being the more highly expressed member. In contrast to the inactivation of individual E2F family members, we have recently demonstrated that loss of Dp1 results in embryonic lethality by embryonic day 12.5 (E12.5) due to the failure of extraembryonic lineages to develop and replicate DNA properly. To bypass this placental requirement and search for roles of Dp1 in the embryo proper, we generated Dp1-deficient embryonic stem (ES) cells that carry the ROSA26-LacZ marker and injected them into wild-type blastocysts to construct Dp1-deficient chimeras. Surprisingly, we recovered mid- to late gestational embryos (E12.5 to E17.5), in which the Dp1-deficient ES cells contributed strongly to most chimeric tissues as judged by X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) staining and Western blotting. Importantly, the abundance of DP2 protein does not increase and the expression of an array of cell cycle genes is virtually unchanged in Dp1-deficient ES cells or chimeric E15.5 tissues with the absence of Dp1. Thus, Dp1 is largely dispensable for embryonic development, despite the absolute extraembryonic requirement for Dp1, which is highly reminiscent of the restricted roles for Rb and cyclins E1/E2 in vivo.

Caspase 3 and 8 deficiency in human neuroblastoma.

An altered apoptotic response represents a pivotal feature of cancer and is involved in cancerogenesis and resistance to chemotherapy. So far, however, only a few studies have been devoted to survey caspase content in malignant cell lines and primary tumor specimens. In this report, we investigated the expression of two pivotal caspases, 3 and 8, in 63 neuroblastoma specimens by three complementary techniques (i.e., reverse transcriptase polymerase chain reaction, immunoblotting, and immunohistochemistry). We confirmed the frequent absence of caspase 8 expression. Moreover and most important, we demonstrated, for the first time to our knowledge, that a significant percentage of neuroblastomas lack caspase 3 mRNA and protein. Both caspase alterations do not show any correlation with tumor stage and MYCN status. Immunohistochemistry showed a large number of caspase-negative cell islets also present in positive samples. Our findings suggest that the absence of caspases might play an important role in neuroblastoma development and resistance to apoptosis-based treatments.

p21Cip1 gene expression is modulated by Egr1: a novel regulatory mechanism involved in the resveratrol antiproliferative effect.

Epidemiological observations indicate that resveratrol, a natural antioxidant stilbene, exerts cardioprotective and chemopreventive effects. Moreover, the molecule induces in vitro cell growth inhibition and differentiation. Using human erythroleukemic K562 cells as model system, we demonstrated that resveratrol induces a remarkable gamma-globin synthesis, the erythroid differentiation being linked to impairment of cell proliferation, increased p21Cip1 expression and inhibition of cdk2 activity. The up-regulation of p21Cip1 transcription is prevented by cycloheximide, indicating the requirement of intermediate protein(s), which, in turn, regulate gene expression. The quantitative analysis of some transcription factors involved in the erythroid lineage, namely GATA-1, GATA-2, and Egr1, indicated that resveratrol selectively up-regulates Egr1 by an Erk1/2-dependent mechanism. The presence of an Egr1 consensus sequence in the p21Cip1 promoter suggested the hypothesis that this transcription factor directly regulates the expression of the cdk inhibitor. Transfection studies with deleted gene promoter constructs, as well as EMSA, pull-down, and chromatin immunoprecipitation experiments substantiated this view, demonstrating that Egr1 binds in vitro and in vivo to the identified consensus sequence of the p21Cip1 promoter. Moreover, an Egr1 phosphorothioate antisense hinders p21Cip1 accumulation and the antiproliferative effects of resveratrol. In conclusion, this is the first demonstration that Egr1 controls p21Cip1 expression by directly interacting with a specific sequence on its gene promoter. The identified regulatory mechanism also contributes to the clarification of the complex chemopreventive and antiproliferative properties of resveratrol.

Antioxidants induce different phenotypes by a distinct modulation of signal transduction.

Antioxidants are known to exert a preventive activity against degenerative diseases. Here, we investigated the mechanism of action of three antioxidants: resveratrol, which causes differentiation of HL-60 cells, and hydroxytyrosol and pyrrolidine dithiocarbamate which, in the same model system, activate apoptosis. The expression profile of hydroxytyrosol-treated cells showed the up-regulation of several genes, including c-jun and egr1. Pyrrolidine dithiocarbamate activates both genes, while resveratrol increases uniquely egr1. A selective modulation of signalling pathway explained this finding. All antioxidants up-regulate Erk1/2, while only hydroxytyrosol and pyrrolidine dithiocarbamate activate c-Jun N-terminal kinase (JNK). Since JNK induces apoptosis by Bcl-2 phosphorylation, we investigated this event. Bcl-2 phosphorylation was increased by hydroxytyrosol and pyrrolidine dithiocarbamate and not by resveratrol. Our results indicate that the different phenotypical effects of antioxidants correlate with modulation of selective transduction pathways.