Differentiated Cell Phenotype Research Articles

Abstract 2237: Involvement of Retinoic Acid Receptors RARβ and RARγ in growth, self-renewal and differentiation on mammary cancer stem cells

Abstract Retinoids exert different effects on cell differentiation and malignant phenotype reversion through the modulation of Retinoid Acid Receptors (RAR). In this work we have analyzed the involvement of retinoid system induction on proliferation, self-renewal and differentiation of cancer stem cells (CSC) using the triple negative mammary cell line LM38-LP. In order to obtain a CSC enriched culture, cells where cultured under mammosphere conditions. We performed mammosphere cultures in order to enrich in CSC. The treatment with All-trans retinoid acid (ATRA1μM for 96 h) reduced CSC growth rate evaluated as mammosphere diameter (Control: 176±8 μm vs. ATRA: 129±10 μm; p≤0.05). Surprisingly, ATRA pre-treatment for 96 h increased CSC self-renewal evaluated as the number of secondary mammospheres (Control: 313±19; ATRA: 495±21; p≤0.05) and as the clonogenic capacity of LM38-LP CSC (Control: 6±0.5; ATRA: 11±1.2; p≤0.05). Concomitant with these biological effects, the same treatment showed, by RT-PCR, an increase in RARβ and RARγ levels in LM38-LP- CSC. Through the use of specific RAR antagonists, we found that RARγ inhibition impaired ATRA effects on self-renewal but RARβ inhibition increased the number of secondary mammospheres (Control: 382±27; ATRA: 735±11; ATRA/RARγ antagonist: 352±17; ATRA/RARβ Antagonist: 1260±177; p≤0.05). A similar result was observed when clonogenic capacity in RARγ blocked CSC was analyzed. Finally, we performed a matrigel 3D culture using cells from LM38-LP mammospheres. Under these conditions, colonies presented large irregular structures while ATRA treatment led to the formation of differentiated colonies with evidence of lumen. Our findings suggest that RARγ down modulation and RARβ induction could lead to CSC self-renewal inhibition and differentiation in triple negative mammary cancers. Citation Format: Damian E. Berardi, Carolina Flumian, Maria I. Diaz Bessone, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Involvement of Retinoic Acid Receptors RARβ and RARγ in growth, self-renewal and differentiation on mammary cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2237. doi:10.1158/1538-7445.AM2015-2237

Abstract LB-267: Comparison of tumor-infiltrating lymphocytes from breast cancer to T cells from adjacent tumor tissue

Abstract While T cells contribute to the eradication of several cancers, including melanoma, the role of T cells present in healthy-appearing, tumor-adjacent tissue is less well described. Memory T cells contain distinct populations of effector memory and central memory, distinguished by their surface phenotype and functions. Effector T cells are thought to be terminally differentiated. We hypothesize that T cells present in healthy-appearing breast tissue contribute to anti-tumor immunity and immune surveillance. Here, we studied breast cancer patients, comparing tumor-infiltrating T cells in breast cancer to T cells from healthy-appearing, tumor-adjacent breast tissue. Since the amount of tissue available for study from excised primary tumors is limited, we utilized a previously established three-dimensional explant method to expand resident T cells from fresh breast tissue. T cell differentiation phenotypes were defined for ten breast cancer specimens and nine tumor-adjacent breast specimens. Six of the tumor/tumor-adjacent tissue specimens were paired. T cell phenotype and function were studied using multiparameter flow cytometry. Tumor-infiltrating CD4 effector T cells (TEFF CD45RO-, CD62L-) were more highly enriched compared to T cells derived from tumor-adjacent breast tissue (mean 21% vs. 6%, p=0.05). In contrast, effector memory CD4 T cells (CD45RO+, CD62L-) were enriched in the healthy adjacent lymphocytes (mean 71% vs. 54%, p<0.05) whereas central memory CD4 T cell (CD45RO+, CD62L+) frequency was equivalent (23% vs. 24%, p=0.83). Functionally, T cells (CD4 and CD8) derived from tumor tissue spontaneously released granules at a higher level than tumor-adjacent T cells as indicated by CD107a expression (1.8% vs 0.8%, p<0.001). In contrast, we detected a higher frequency of mitogen-induced CCL4-producing CD4 T cells from tumor-adjacent tissue vs. tumor (62% vs. 51%, p<0.05), as well as a higher frequency of IFN-γ-producing CD8 T cells (80% vs. 65%, p<0.05). These studies demonstrate the feasibility of using this three-dimensional explant approach to investigate the role of T cells in immune surveillance against breast cancer. Our findings indicate that T cell phenotypes differ in breast tissue; tumor-adjacent T cells are enriched in effector memory cells whereas TILs are characterized by terminally differentiated effector T cells. The data further suggest that breast TILs, through their comparatively reduced frequency of CCL4- and IFN-γ-producing cells have a diminished functional capacity to restrict tumor growth and metastasis. Citation Format: Yi Guo, Jaime Shamonki, Michelle Kinnaird, Maggie DiNome, Maureen Chung, Peter Sieling, Delphine J. Lee. Comparison of tumor-infiltrating lymphocytes from breast cancer to T cells from adjacent tumor tissue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-267. doi:10.1158/1538-7445.AM2014-LB-267

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

Stabilizing hepatocellular phenotype using optimized synthetic surfaces.

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

Real-time discrimination between proliferation and neuronal and astroglial differentiation of human neural stem cells.

Neural stem cells (NSCs) are characterized by a capacity for self-renewal, differentiation into multiple neural lineages, all of which are considered to be promising components for neural regeneration. However, for cell-replacement therapies, it is essential to monitor the process of in vitro NSC differentiation and identify differentiated cell phenotypes. We report a real-time and label-free method that uses a capacitance sensor array to monitor the differentiation of human fetal brain-derived NSCs (hNSCs) and to identify the fates of differentiated cells. When hNSCs were placed under proliferation or differentiation conditions in five media, proliferating and differentiating hNSCs exhibited different frequency and time dependences of capacitance, indicating that the proliferation and differentiation status of hNSCs may be discriminated in real-time using our capacitance sensor. In addition, comparison between real-time capacitance and time-lapse optical images revealed that neuronal and astroglial differentiation of hNSCs may be identified in real-time without cell labeling.

Involvement of protein kinase C α and δ activities on the induction of the retinoic acid system in mammary cancer cells.

It has been established that retinoids exert some of their effects on cell differentiation and malignant phenotype reversion through the interaction with different members of the protein kinase C (PKC) family. Till nowadays the nature and extension of this interaction is not well understood. Due to the cytostatic and differentiating effects of retinoids, in the present study we propose to evaluate whether the crosstalk between the retinoid system and the PKC pathway could become a possible target for breast cancer treatment. We could determine that ATRA (all-trans retinoic) treatment showed a significant growth inhibition due to (G1 or G2) cell cycle arrest both in LM3 and SKBR3, a murine and human mammary cell line respectively. ATRA also induced a remarkable increase in PKCα and PKCδ expression and activity. Interestingly, the pharmacological inhibition of these two PKC isoforms prevented the activation of retinoic acid receptors (RARs) by ATRA, indicating that both PKC isoforms are required for RARs activation. Moreover, PKCδ inhibition also impaired ATRA-induced RARα translocation to the nucleus. In vivo assays revealed that a combined treatment using ATRA and PKCα inhibitors prevented lung metastatic dissemination in an additive way. Our results clearly indicate that ATRA modulates the expression and activity of different PKCs. Besides inducing cell arrest, the activity of both PKC is necessary for the induction of the retinoic acid system. The combined ATRA and PKCα inhibitors could be an option for the hormone-independent breast cancer treatment.

MiR-1247 Functions by Targeting Cartilage Transcription Factor SOX9

microRNAs are a large and essential class of gene regulators that play key roles in development, homeostasis, and disease. They are necessary for normal skeletal development, and their expression is altered in arthritis. However, the specific role of individual microRNAs is only beginning to be unraveled. Using microRNA expression profiling in healthy human articular cartilage cells (chondrocytes), we identified miR-1247 expression as highly correlated with that of the differentiated cell phenotype. Transcribed from the DLK1-DIO3 locus, the function of miR-1247 is completely unknown. In mice its expression level was relatively high in cartilage tissue, and correlated with cartilage-associated microRNA miR-675 across a range of 15 different mouse tissues. To further probe miR-1247 function, overexpression and inhibition studies were performed in isolated human chondrocytes. Modulation of miR-1247 was found to exert profound phenotypic effects altering expression levels of cartilage master regulator transcription factor SOX9. SOX9 is essential for cartilage development and subsequent function throughout life, and mutations in this gene result in severe dwarfism. Putative miR-1247 binding sites were further investigated using luciferase reporter assays, which indicated binding of miR-1247 to a highly conserved region in the coding sequence of SOX9 but not in its 3'-UTR. Interestingly, depletion of SOX9 in human chondrocytes resulted in increased levels of the mature, processed microRNA, suggesting a negative feedback loop between miR-1247 and its target SOX9.

Induce and differenctiation of marrow mesenchymal stem cell into photoreceptor-like cell in vitro and microenvironment

Background Marrow mesenchymal stem cells （MSCs） has been successful induced to differentiate into corneal cells,retinal ganglion cells （RGCs） and retinal neuron-like cells in recent years, but there are few studies about MSCs induced into photoreceptor cells and their microenvironment. Objective The aim of this study was to explore the induce and differentiation of bone marrow mesenehymal stem cells （BMSCs） into photoreeeptor-like cells in vitro and mieroenvironment. Methods The second generation of human BMSCs strain and RPE cells strain were cultured and passaged, respectively, and the fourth generation of BMSCs and the third generation of RPE cells were used in the experiment. BMSCs were eoeuhured using the mesenchymal stem cells medium （MSCM） contained 20 μg/L basic fibroblast growth factor （bFGF） ,20 μg/L epithelial growth factor （EGF） and 20μg/L brain derived neurotrophie factor （BDNF） with RPE cells to induce the differentiation of BMSCs in the induced group, and BMSCs were cultured in MSCM only in the control group. The morphology of induced and differentiated cells were observed under the inverted microscope. Inmmunocytoehemistry was used in induced for 3-, 5-,7-day cells to detect the expression rate of rhodopsin protein for the identification of phenotype of the differentiated cells. RT-PCR was used in induced for 5-,7-day cells to detect the expressions of rhodopsin mRNA and recoverin mRNA. Results Cultured BMSCs grew well with the spindle shape,and passaged RPE cells showed the uniform size and polygon shape with the abundant pigment in the cells. Some induced cells appeared to be the neuron-like cells with round shape and long prominence and the secondary reticular branches. The expression rates of rhodopsinin the cells were （5.83±0.29）%, （20. 36±0. 32）% and （29.80±2. 30）% in the third,fifth and seventh day after induce, which were significantly higher than （0.71 ±0. 35 ） % , （ 2. 56±0.24 ） % and （ 2. 32±0. 42 ） % of control cells （ t3 d = 41. 510, t5 d = 107. 290, t7 d = 30. 036, P〈0. 01 ）. The grey scales of rhodopsin mRNA and recoverin mRNA were significantly elevated in the induced and differentiated cells compared with control cells in the fifth and seventh day （ rhodopsin mRNA ：t5 d = 103. 506, t7 d = 122. 584, P〈0. 01 ;reeoverin mRNA ：t5 d = 106. 674, t7 d = 189. 992 ,P〈0. 01 ）. Conclusions BMSCs can be successfully indueed to differentiate into photoreeeptor cells after cocultured by conditioned medium with RPE cells. Key words: Mesenehymal stem cell; Bone marrow; Indueed and differentiation; Photoreceptor-like cell; Mieroenvironment; Conditioned medium; Rhodopsin; Reeoverin

Abstract 555: Proliferation, apoptosis and senescence differential modulation by combining the retinoid ATRA and Protein Kinase C pharmacological inhibitors, in a bicellular murine mammary tumor cell line.

Abstract Retinoids exert some of their effects on cell differentiation and malignant phenotype reversion through interactions with specific Protein Kinase C (PKC) isoforms. In this work we studied: A)- the modulation of PKCα and PKCδ expression when cells were treated with ATRA (1 μM), B)- The expression profile of Retinoic Acid Receptors (RAR) when cells are treated with ATRA and/or PKCα / PKC δ inhibitors (Gö6976 2.5 μM / Rottlerin 1 μM) and C)- the effect of combining ATRA and PKC inhibitors on in vitro cell growth, cell cycle distribution and senescence. We used as model the murine mammary tumor-derived cell line LM38-LP, composed by luminal (LEP) and myoepithelial (MEP) cells. By RT-PCR we could determine that 48 h ATRA treatment increased PKCδ only in the LM38-LP LEP population and decreased PKCα expression levels in both cellular components. Regarding RARs profile, the treatment with PKCα inhibitor Gö6976 reduced RARγ2 levels, while its combination with ATRA also increased RARβ2. Furthermore, 96 h treatment with ATRA, Gö6976, or Rottlerin or their combinations induced cell growth inhibition (cell number x105: control 22± 1.1; ATRA 11±0.1; Gö6976 3.6±0.6; Rottlerin 9±0.1; ATRA/Gö6976 2.3±0.03; ATRA/Rottlerin 6.7±1.3, p ≤0.05 vs control in all treatments). By flow cytometry we could determine that both ATRA and Rottlerin treatments induce LM38-LP cell cycle arrest in G1 phase, while Gö6976 increased the number of cells in the subG1 phase, correlating with increased apoptosis and confirmed by Annexin V-FITC staining. Employing β-galactosidase activity assay and bright field microscopy, we determined that 96 h ATRA treatment induced senescence mainly in the MEP component while Gö6976 does it in the LEP population. Interestingly, Rottlerin treated cells showed morphological alterations compatible with autophagy. We conclude that cell growth inhibition exerted by retinoids may be enhanced by the reduction in PKCα expression and / or activity. In addition the combination of ATRA plus Gö6976, induced differential responses on the different LM38-LP cell components, showing senescence in MEP cells, together with cell cycle arrest and apoptosis mainly in the LEP compartment. Consistently, all these biological effects correlated with an increase in RARβ2 (known to induce cell differentiation) and a decrease in RARγ2 (implicated in proliferation) mRNAs level. Citation Format: Damian E. Berardi, Maria I. Diaz Bessone, Carolina Flumian, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Proliferation, apoptosis and senescence differential modulation by combining the retinoid ATRA and Protein Kinase C pharmacological inhibitors, in a bicellular murine mammary tumor cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 555. doi:10.1158/1538-7445.AM2013-555

Abstract 3015: Crosstalk between ≤ and ≤ PKC isoforms and retinoic acid system in malignant phenotype reversion

Abstract Retinoids may exert some of their effects on cell differentiation and malignant phenotype reversion through interaction with different PKC isoforms. In this work we studied the crosstalk between retinoid acid system (RA) and PKC signaling pathway and its implication in malignant phenotype reversion using a murine mammary tumor cell line (LM3) and a human breast cancer-derived cell line (MDA-MB231). ATRA (all trans RA) treatment (1uM,72h) induced a significant growth inhibition in vitro in LM3 cell line. This result was confirmed in vivo when LM3 cells were injected orthotopically in female BALB/c mice while carrying subcutaneous slow release ATRA pellets (10 mg). This phenomenon was associated with the reduction of pErk1 levels (80±9%) and the increase of the cell cycle inhibitor p27 in the nuclear fraction without altering Cyclin D1 expression in LM3 cell line. None of these modulations were observed in ATRA unresponsive MDA-MB231 cell line. We determined by Western blot that 24h treatment with ATRA induced an increase of PKC∈ and PKC∈ levels, detecting alpha isoform only in the membrane fraction and delta isoform in the nuclear fraction. In contrast, in MDA-MB231 cells, PKC∈ and PKC∈ expression was reduced after ATRA exposure. Interestingly, pharmacological inhibition of PKC∈ and PKC∈ prevented retinoid receptors activation by ATRA in LM3 cells, as evidenced by a reporter gene assay (RARE-Luciferase). Western blot showed that pharmacological inhibition of PKC∈ (Rottlerin 1μM, 24h) impaired ATRA-induced RARα1 translocation to the nucleus while the pharmacological inhibition of PKC∈ (Gö6976 5μM, 24h) did not affect this translocation. Moreover, immunoprecipitation assays showed that only PKC∈ co-immunoprecipitated with RARα1 after ATRA treatment, suggesting a physical interaction between both molecules. Rottlerin-ATRA treatment (72h) reversed the effect on proliferation rate exerted by retinoid treatment. Gö6976-ATRA treatment (72h) significantly decreased cell duplication rate compared with ATRA or Gö6976 treatment alone, in an additive manner. Pre-treated LM3 cells with Gö6976 were intravenously injected in female BALB/c mice that carried subcutaneous slow release ATRA pellets in order to evaluate the importance of combination therapy in the last stages of metastatic spread. We noticed that this combined therapy produced a higher decrease in the number of lung metastases, suggesting an additive effect. Our results indicate that PKC/Retinoid Crosstalk is implicated in growth inhibition through the induction of RA transcription genes that lead to cell cycle arrest and differentiation. Finally we speculate that retinoid treatment combined with PKC∈ isoform modulators could be considered for treatment of PKC∈ positive breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3015. doi:1538-7445.AM2012-3015

Abstract 1654: The low-penetrance breast cancer risk allele MCS5A functions in T-cells through downregulation of the E3-ubiquitin ligase Fbxo10, to alter mammary epithelial cell differentiation

Abstract Genome-wide association studies (GWAS) have identified ∼50 low-penetrance, high-population frequency breast cancer susceptibility variants, of which the vast majority are non-protein coding. Mechanisms through which such variants modulate breast cancer risk are largely unknown. The non-coding mammary carcinoma susceptibility locus 5a (MCS5A/Mcs5a) is associated with breast cancer risk in both women and rats (Samuelson et al., 2007 PNAS). We use a comparative genetics approach to functionally characterize this locus. Using mammary gland and bone marrow transplantation assays in congenic rat models, we showed that the Mcs5a resistance allele acts through a non-mammary cell-autonomous immune mechanism. We found that Mcs5a is associated with altered T-cell homeostasis and functions, most notably a 38% and 89% higher abundance of gammadelta T-cells in the spleen and mammary gland, respectively (Smits et al., 2011 Breast Cancer Res). On the molecular genetic level, the non-protein coding Mcs5a resistance allele was found to strongly associate with ∼2-fold and ∼4-fold lower Fbxo10 transcript levels in thymic and splenic T-cells, respectively, but not in other immune cell types or mammary gland (Smits et al., 2011 Nucleic Acids Res). Fbxo10 is an E3-ubiquitin ligase gene, transcriptionally initiating from within Mcs5a. Using flow cytometric analysis of the rat mammary epithelium with antibodies against CD24, CD29, CD31, CD45, cytokeratin (CK) 14, CK19, as well as staining with phalloidin, we identified the luminal and basal/myoepithelial cell populations (Sharma et al., 2011 PLoS ONE). Quantitative analysis of these populations revealed that in a window of cancer susceptibility to carcinogen exposure (∼50-90 days of age), mammary glands from Mcs5a resistant congenic rats have a 47% lower frequency of basal/myoepithelial (CD24medCD29high) cells. Loss of CD29 (Integrin-beta1) expression in the mouse mammary epithelium has been implicated in mammary cancer resistance. To investigate the function of Fbxo10, we generated an Fbxo10 knockout mouse model, which recapitulated both the gammadelta T-cell and the mammary epithelial cell differentiation phenotypes. We hypothesize that Mcs5a controls Fbxo10 expression levels in the thymus, resulting in higher abundance of gammadelta T-cells, especially in the mammary epithelium, which contributes to resistance to carcinoma development through increased immune surveillance and/or altered mammary epithelial cell differentiation. Fbxo10 and its downstream ubiquitination proteins represent novel breast cancer prevention targets within the immune system. This study exemplifies the necessity of mammalian genetic models for deciphering mechanisms through which low-penetrance variants affect cancer susceptibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1654. doi:1538-7445.AM2012-1654

Comparing predictive drug nephrotoxicity biomarkers in kidney 3-D primary organoid culture and immortalized cell lines

The cellular microenvironment is recognized to play a key role in stabilizing cell differentiation states and phenotypes in culture. This study addresses the hypothesis that preservation of in vivo-like tissue architecture in vitro produces a cell culture more capable of responding to environmental stimuli with clinically relevant toxicity biomarkers. This was achieved using kidney proximal tubules in three-dimensional organoid hydrogel culture, with comparisons to conventional monolayer kidney cell cultures on plastic. Kidney proximal tubule cultures and two immortalized kidney cell line monolayer cultures exposed to known nephrotoxic drugs were evaluated for inflammatory cytokines, nephrotoxicity-associated genes, Kim-1 protein, cytochrome enzymes, and characteristic cellular enzyme shedding. Significant similarities are shown for these traditional biomarkers of kidney toxicity between in vivo and 3-D organoid endpoints of drug toxicity, and significantly, a consistent lack of clinically relevant endpoints produced by traditional 2-D kidney cell cultures. These findings impact both in vitro bioreactor-based kidney functional and regenerative medicine models, as well as high-throughput cell-based drug screening validations.

Generation of mouse ES cell lines engineered for the forced induction of transcription factors

Here we report the generation and characterization of 84 mouse ES cell lines with doxycycline-controllable transcription factors (TFs) which, together with the previous 53 lines, cover 7–10% of all TFs encoded in the mouse genome. Global gene expression profiles of all 137 lines after the induction of TFs for 48 hrs can associate each TF with the direction of ES cell differentiation, regulatory pathways, and mouse phenotypes. These cell lines and microarray data provide building blocks for a variety of future biomedical research applications as a community resource.

Expression of Germinal Center B Cell Markers in Bortezomib-Resistant Multiple Myeloma Cells

Abstract 129Multiple myeloma (MM), the second most common hematopoietic malignancy, is an incurable plasma cell neoplasm representing approximately 2% of all cancer deaths. The proteasome inhibitor, bortezomib (Bz), has been widely used to treat MM, and emerging next-generation proteasome inhibitors are being evaluated. Despite advances in therapies, patients treated with Bz eventually relapse due to the development of drug resistance. Thus, identifying signatures that distinguish Bz sensitive from resistant MM cells will more accurately predict Bz sensitivity and will aid in the development of novel approaches to reverse resistance.MM populations are characterized by phenotypic heterogeneity suggesting the possibility that certain MM subpopulations are associated with Bz resistance. In order to characterize these populations in greater detail, we have utilized isogenic pairs of Bz sensitive and resistant MM lines derived from the genetically engineered iMycCa/Bcl-xl mouse model of plasma cell malignancy. The iMycCa/Bcl-xl mouse is a highly robust model that very closely resembles human MM based on gene expression profiling (GEP), chromosomal abnormalities and progression of disease and response to therapy (Boylan, et al. Cancer Res (2007) 67: 4069). We have employed this mouse model because of its striking similarity to human MM, and MM cells isolated from these mice can be readily infected by lentivirus as well as adoptively transferred into syngeneic, immunocompetent recipients establishing a powerful system to directly test biologically relevant hypotheses relating to Bz resistance.Isogenic pairs of Bz sensitive and resistant lines were extensively characterized using GEP, quantitative PCR and flow cytometry. We observed increased expression of germinal B cell markers Pax5, AID and Bcl-6 and reduced expression of the plasma cell marker CD93 in Bz resistant lines compared to Bz sensitive pairs suggesting that Bz resistance is associated with germinal center-like B cell characteristics. Remarkably, we detect within the same cell co-expression of both CD138/Pax5 and CD138/AID demonstrating the tremendous plasticity of these cells and their intermediate B cell differentiation phenotype. Recently, both AID and Bcl-6 have been identified as crucial mediators of resistance to tyrosine kinase inhibitors in leukemia cells (Duy, et al. Nature (2011) 473: 384; Klemm, et al. Cancer Cell (2009) 16: 232) raising an intriguing possibility that these proteins thought to be restricted to germinal center B cells could play a role in Bz resistance in MM. These observations have led us to more closely characterize the apparent shift in developmental stage resulting from Bz-driven expression changes or selection. Time course analysis of gene and protein expression following low-dose Bz treatment in the absence of cell death indicates that Bz increases the expression and promotes the stabilization of Pax5, AID and Bcl-6. This demonstrates a drug-induced change in markers associated with B cell differentiation relating to the ultimate emergence of the resistance phenotype. We are now determining whether similar B cell differentiation phenotypes emerge in a recently published human drug trial where GEP was completed prior to and 48 hours after Bz treatment in patients (Shaughnessy, et al. Blood (2011), ahead of print). Thus, we will present phenotypic signatures correlating to Bz resistance in cell lines derived from our mouse model of MM and suggest that targeting B cell differentiation may represent a rational therapeutic approach to overcoming Bz resistance. Disclosures:Stessman:Millennium: The Takeda Oncology Company: Research Funding. Mansoor:Millennium: The Takeda Oncology Company: Research Funding. Van Ness:Millennium: The Takeda Oncology Company: Research Funding.

Nitric oxide causes root apical meristem defects and growth inhibition while reducing PIN-FORMED 1 (PIN1)-dependent acropetal auxin transport

Nitric oxide (NO) is considered a key regulator of plant developmental processes and defense, although the mechanism and direct targets of NO action remain largely unknown. We used phenotypic, cellular, and genetic analyses in Arabidopsis thaliana to explore the role of NO in regulating primary root growth and auxin transport. Treatment with the NO donors S-nitroso-N-acetylpenicillamine, sodium nitroprusside, and S-nitrosoglutathione reduces cell division, affecting the distribution of mitotic cells and meristem size by reducing cell size and number compared with NO depletion by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Interestingly, genetic backgrounds in which the endogenous NO levels are enhanced [chlorophyll a/b binding protein underexpressed 1/NO overproducer 1 (cue1/nox1) mirror this response, together with an increased cell differentiation phenotype. Because of the importance of auxin distribution in regulating primary root growth, we analyzed auxin-dependent response after altering NO levels. Both elevated NO supply and the NO-overproducing Arabidopsis mutant cue1/nox1 exhibit reduced expression of the auxin reporter markers DR5pro:GUS/GFP. These effects were accompanied by a reduction in auxin transport in primary roots. NO application and the cue1/nox1 mutation caused decreased PIN-FORMED 1 (PIN1)-GFP fluorescence in a proteasome-independent manner. Remarkably, the cue1/nox1-mutant root phenotypes resemble those of pin1 mutants. The use of both chemical treatments and mutants with altered NO levels demonstrates that high levels of NO reduce auxin transport and response by a PIN1-dependent mechanism, and root meristem activity is reduced concomitantly.

New Simple and Rapid Method for Purification of Mesenchymal Stem Cells from the Human Umbilical Cord Wharton Jelly

We have developed a simple and rapid method for isolation of human umbilical cord matrix stem cells (hUCMS). The umbilical cord contains a virtual inexhaustible source of adult stem cells. We have substantially modified, simplified, and improved previously reported hUCMS isolation procedures in terms of either used enzyme type, or digestion time, and substantially enhanced the final product yield and purity. The isolated hUCMS were positive for CD90, CD117, and SCF, and negative for CD31 and CD45 surface markers. mRNA and related proteins (i.e., Sox2, Oct4a, Nanog, ABCG2, and c-Myc) that coincide with an uncommitted cell status also were detected. hUCMS express genes and proteins for CD90 and Nestin that are associated with mesenchymal stem cells, as well as other genes that specifically relate to different embryonic germ layers, namely, Vimentin, Sox7, Sox17, FoxA2, E-cadherin, and N-cadherin. hUCMS showed multilineage cell differentiation potential into adipogenic, osteogenic, and neural cell phenotypes, under the influence of lineage-specific, differentiation culture media. Moreover, the basal expression of endocrine cell markers makes these cells seemingly suitable for endocrine cell phenotype differentiation. Noteworthy, Activin A induced hUCMS to acquire definitive endoderm cell markers.

Abstract 3126: Intercellular communication in the urothelium: Evidence for a switch in gap junction regulation during differentiation

Abstract Cells have a requirement for intercellular communication in order to maintain normal tissue polarity and homeostasis. Knowledge of the signaling molecules transferred through specific gap junctions is limited; however aberrant expression of connexins, the protein subunits of gap junctions, has been linked to many disorders including neoplastic transformation of brain and colorectal tissues. A number of reports have documented changes in connexin expression associated with bladder cancer. The aims of this study were 1) to define the connexins expressed by the epithelial lining of the human urinary tract (urothelium) in situ and in vitro; 2) to determine if expressed connexins assemble into functional gap junctions and 3) the contribution of specific connexins to urothelial tissue physiology. Established culture systems for normal human urothelial (NHU) cells were used to replicate proliferative (regenerative) or differentiated cell phenotypes. RT-PCR was used to screen normal urothelial tissue and NHU cell cultures for expression of connexin genes. Immunochemical techniques were employed to determine connexin expression and localization in situ and in vitro. Dye-transfer techniques were used on cultured cells to confirm that assembled gap junctions supported functional cell-cell communication. Of the 20 human connexin genes, six were expressed in situ and by differentiated cultures, whilst two were expressed only in proliferative culture conditions. NHU cells were capable of dye transfer in both proliferative and differentiated cultures, indicating the presence of functional gap junctions. Immunofluorescence microscopy demonstrated that the switch in medium from a low (0.09mM) to near physiological (2mM) calcium concentration resulted in increased assembly of gap junctions containing connexin-43. Connexin-32 was only expressed by differentiated cultures, as shown by RT-PCR and immunochemistry, where it was localized to intercellular borders indicating a functional role. This data represents the first systematic characterization of connexin family expression in the urothelium and provides evidence that urothelial cells alter their connexin expression upon differentiation to produce specific channels. Connexin-32 has been reported to be involved in terminal differentiation of human mammary gland and future work will focus on characterizing the role of connexins in defining the differentiated urothelial phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3126. doi:10.1158/1538-7445.AM2011-3126

Abstract 1199: Implications of protein kinase C (PKC)α and PKCΔ on murine mammary tumor growth and metastatic dissemination; effect of retinoids

Abstract The retinoid system exerts some of its effects on cell differentiation and malignant phenotype reversion through the interaction of its receptors (RAR) with different PKC isoforms. In this work, using a murine mammary tumor cell line (LM3), we studied the interaction between RARs and PKC in vitro as well as whether the overexpression of α and Δ PKC isoforms could influence the effect of retinoids on cell proliferation both in vivo and in vitro. Western blot assays showed that the pharmacological inhibition of PKCΔ (Rottlerin 5μM, 24h) impaired all trans retinoic acid (ATRA)-induced RARα1 translocation to the nucleus while the pharmacological inhibition of PKCα (Gö6976 5μM, 24h) did not affect this translocation. Moreover, immunoprecipitation assays showed that only PKCΔ co-immunoprecipitated with RARα1 after ATRA treatment, suggesting a physical interaction between both molecules. However, both PKC isoforms seemed to be necessary for RARs activation as determined by a gene reporter assay. Next, by a stable transfection procedure, we overexpressed PKCα and PKCΔ in LM3 cell line. In vitro assays showed that LM3-PKCα cells presented the highest proliferative rate and were highly responsive to ATRA treatment reducing their growth capability. These genetically modified lines were also inoculated orthotopically into the mammary glands of female BALB/c mice. Ten days later, when tumors were palpable, mice were implanted with ATRA pellets (10 mg/mouse). Tumors overexpressing PKCα presented a higher growth rate and were more metastatic than control vector-transfected LM3 cells, but showed a significant response to ATRA treatment, impairing primary tumor growth and reducing lung metastases number [Md (range) 55 (20-75) vs. 16 (0-27) in non-treated and ATRA-treated LM3-PKCα cells respectively (P<0.05 Mann-Whitney test)]. Both control and PKCΔ overexpressing tumors were unresponsive to retinoid treatment.We can conclude that, while PKCα overexpressing cells were more aggressive than control ones, they showed an important response to retinoid treatment both in vivo and in vitro, indicating that a retinoid therapy could be successful in human mammary tumors with high PKCα levels. On the other hand, while we found an in vitro clear interaction between PKCΔ and RARα1, the overexpression of PKCΔ did not to modulate their response to the retinoid therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1199. doi:10.1158/1538-7445.AM2011-1199

Eya1 controls cell polarity, spindle orientation, cell fate and Notch signaling in distal embryonic lung epithelium

Cell polarity, mitotic spindle orientation and asymmetric division play a crucial role in the self-renewal/differentiation of epithelial cells, yet little is known about these processes and the molecular programs that control them in embryonic lung distal epithelium. Herein, we provide the first evidence that embryonic lung distal epithelium is polarized with characteristic perpendicular cell divisions. Consistent with these findings, spindle orientation-regulatory proteins Insc, LGN (Gpsm2) and NuMA, and the cell fate determinant Numb are asymmetrically localized in embryonic lung distal epithelium. Interfering with the function of these proteins in vitro randomizes spindle orientation and changes cell fate. We further show that Eya1 protein regulates cell polarity, spindle orientation and the localization of Numb, which inhibits Notch signaling. Hence, Eya1 promotes both perpendicular division as well as Numb asymmetric segregation to one daughter in mitotic distal lung epithelium, probably by controlling aPKCζ phosphorylation. Thus, epithelial cell polarity and mitotic spindle orientation are defective after interfering with Eya1 function in vivo or in vitro. In addition, in Eya1(-/-) lungs, perpendicular division is not maintained and Numb is segregated to both daughter cells in mitotic epithelial cells, leading to inactivation of Notch signaling. As Notch signaling promotes progenitor cell identity at the expense of differentiated cell phenotypes, we test whether genetic activation of Notch could rescue the Eya1(-/-) lung phenotype, which is characterized by loss of epithelial progenitors, increased epithelial differentiation but reduced branching. Indeed, genetic activation of Notch partially rescues Eya1(-/-) lung epithelial defects. These findings uncover novel functions for Eya1 as a crucial regulator of the complex behavior of distal embryonic lung epithelium.

Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots

Brassinosteroids (BRs) play crucial roles in plant growth and development. Previous studies have shown that BRs promote cell elongation in vegetative organs in several plant species, but their contribution to meristem homeostasis remains unexplored. Our analyses report that both loss- and gain-of-function BR-related mutants in Arabidopsis thaliana have reduced meristem size, indicating that balanced BR signalling is needed for the optimal root growth. In the BR-insensitive bri1-116 mutant, the expression pattern of the cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE revealed that a decreased mitotic activity accounts for the reduced meristem size; accordingly, this defect could be overcome by the overexpression of CYCD3;1. The activity of the quiescent centre (QC) was low in the short roots of bri1-116, as reported by cell type-specific markers and differentiation phenotypes of distal stem cells. Conversely, plants treated with the most active BR, brassinolide, or mutants with enhanced BR signalling, such as bes1-D, show a premature cell cycle exit that results in early differentiation of meristematic cells, which also negatively influence meristem size and overall root growth. In the stem cell niche, BRs promote the QC renewal and differentiation of distal stem cells. Together, our results provide evidence that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem.