Tumor Cell-Intrinsic c-Myb Upregulation Stimulates Antitumor Immunity in a Murine Colorectal Cancer Model.

The transcription factor c-Myb is overexpressed in many different types of solid tumors, including colorectal cancer. However, its exact role in tumorigenesis is unclear. In this study, we show that tumor-intrinsic c-Myb expression in mouse models of colon cancer and melanoma suppresses tumor growth. Although no differences in proliferation, apoptosis, and angiogenesis of tumors were evident in tumors with distinct levels of c-Myb expression, we observed changes in intratumoral immune cell infiltrates. MC38 tumors with upregulated c-Myb expression showed increased numbers of CD103+ dendritic cells and eosinophils, but decreased tumor-associated macrophages (TAM). Concomitantly, an increase in the number of activated cytotoxic CD8+ T cells upon c-Myb upregulation was observed, which correlated with a pro-inflammatory tumor microenvironment and increased numbers of M1 polarized TAMs. Mechanistically, c-Myb upregulation in immunogenic MC38 colon cancer cells resulted in enhanced expression of immunomodulatory genes, including those encoding ß2-microglobulin and IFNß, and decreased expression of the gene encoding the chemokine receptor CCR2. The increased numbers of activated cytotoxic CD8+ T cells contributed to tumor growth attenuation. In poorly immunogenic CT26, LLC, and B16-BL6 tumor cells, c-Myb upregulation did not affect the immunomodulatory gene expression. Despite this, c-Myb upregulation led to reduced B16-BL6 tumor growth but it did not affect tumor growth of CT26 and LLC tumors. Altogether, we postulate that c-Myb functions as a tumor suppressor in a tumor cell-type specific manner and modulates antitumor immunity.

TACSTD2 upregulation is an early reaction to lung infection.

TACSTD2 encodes a transmembrane glycoprotein Trop2 commonly overexpressed in carcinomas. While the Trop2 protein was discovered already in 1981 and first antibody-drug conjugate targeting Trop2 were recently approved for cancer therapy, the physiological role of Trop2 is still not fully understood. In this article, we show that TACSTD2/Trop2 expression is evolutionarily conserved in lungs of various vertebrates. By analysis of publicly available transcriptomic data we demonstrate that TACSTD2 level consistently increases in lungs infected with miscellaneous, but mainly viral pathogens. Single cell and subpopulation based transcriptomic data revealed that the major source of TACSTD2 transcript are lung epithelial cells and their progenitors and that TACSTD2 is induced directly in lung epithelial cells following infection. Increase in TACSTD2 expression may represent a mechanism to maintain/restore epithelial barrier function and contribute to regeneration process in infected/damaged lungs.

Transcription factor c-Myb: novel prognostic factor in osteosarcoma.

The transcription factor c-Myb is an oncoprotein promoting cell proliferation and survival when aberrantly activated/expressed, thus contributing to malignant transformation. Overexpression of c-Myb has been found in leukemias, breast, colon and adenoid cystic carcinoma. Recent studies revealed its expression also in osteosarcoma cell lines and suggested its functional importance during bone development. However, the relevance of c-Myb in control of osteosarcoma progression remains unknown. A retrospective clinical study was carried out to assess a relationship between c-Myb expression in archival osteosarcoma tissues and prognosis in a cohort of high-grade osteosarcoma patients. In addition, MYB was depleted in metastatic osteosarcoma cell lines SAOS-2 LM5 and 143B and their growth, chemosensitivity, migration and metastatic activity were determined. Immunohistochemical analysis revealed that high c-Myb expression was significantly associated with poor overall survival in the cohort and metastatic progression in young patients. Increased level of c-Myb was detected in metastatic osteosarcoma cell lines and its depletion suppressed their growth, colony-forming capacity, migration and chemoresistance in vitro in a cell line-dependent manner. MYB knock-out resulted in reduced metastatic activity of both SAOS-2 LM5 and 143B cell lines in immunodeficient mice. Transcriptomic analysis revealed the c-Myb-driven functional programs enriched for genes involved in the regulation of cell growth, stress response, cell adhesion and cell differentiation/morphogenesis. Wnt signaling pathway was identified as c-Myb target in osteosarcoma cells. Taken together, we identified c-Myb as a negative prognostic factor in osteosarcoma and showed its involvement in the regulation of osteosarcoma cell growth, chemosensitivity, migration and metastatic activity.

c-Myb interferes with inflammatory IL1α-NF-κB pathway in breast cancer cells.

The transcription factor c-Myb can be involved in the activation of many genes with protumorigenic function; however, its role in breast cancer (BC) development is still under discussion. c-Myb is considered as a tumor-promoting factor in the early phases of BC, on the other hand, its expression in BC patients relates to a good prognosis. Previously, we have shown that c-Myb controls the capacity of BC cells to form spontaneous lung metastasis. Reduced seeding of BC cells to the lungs is linked to high expression of c-Myb and a decline in expression of a specific set of inflammatory genes. Here, we unraveled a c-Myb-IL1α-NF-κB signaling axis that takes place in tumor cells. We report that an overexpression of c-Myb interfered with the activity of NF-κB in several BC cell lines. We identified IL1α to be essential for this interference since it was abrogated in the IL1α-deficient cells. Overexpression of IL1α, as well as addition of recombinant IL1α protein, activated NF-κB signaling and restored expression of the inflammatory signature genes suppressed by c-Myb. The endogenous levels of c-Myb negatively correlated with IL1α on both transcriptional and protein levels across BC cell lines. We concluded that inhibition of IL1α expression by c-Myb reduces NF-κB activity and disconnects the inflammatory circuit, a potentially targetable mechanism to mimic the antimetastatic effect of c-Myb with therapeutic perspective.

Large-Scale Automated Hollow-Fiber Bioreactor Expansion of Umbilical Cord-Derived Human Mesenchymal Stromal Cells for Neurological Disorders.

Neurodegenerative disorders present a broad group of neurological diseases and remain one of the greatest challenges and burdens to mankind. Maladies like amyotrophic lateral sclerosis, Alzheimer's disease, stroke or spinal cord injury commonly features astroglia involvement (astrogliosis) with signs of inflammation. Regenerative, paracrine and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) could target the above components, thus opening new therapeutic possibilities for regenerative medicine. A special interest should be given to hMSCs derived from the umbilical cord (UC) tissue, due to their origin, properties and lack of ethical paradigms. The aim of this study was to establish standard operating and scale-up good manufacturing practice (GMP) protocols of UC-hMSCs isolation, characterization, expansion and comparison of cells' properties when harvested on T-flasks versus using a large-scale bioreactor system. Human UC-hMSCs, isolated by tissue explant culture technique from Wharton's jelly, were harvested after reaching 75% confluence and cultured using tissue culture flasks. Obtained UC-hMSCs prior/after the cryopreservation and after harvesting in a bioreactor, were fully characterized for "mesenchymness" immunomodulatory, tumorigenicity and genetic stability, senescence and cell-doubling properties, as well as gene expression features. Our study demonstrates an efficient and simple technique for large scale UC-hMSCs expansion. Harvesting of UC-hMSCs' using classic and large scale methods did not alter UC-hMSCs' senescence, genetic stability or in vitro tumorigenicity features. We observed comparable growth and immunomodulatory capacities of fresh, frozen and expanded UC-hMSCs. We found no difference in the ability to differentiate toward adipogenic, osteogenic and chondrogenic lineages between classic and large scale UC-hMSCs expansion methods. Both, methods enabled derivation of genetically stabile cells with typical mesenchymal features. Interestingly, we found significantly increased mRNA expression levels of neural growth factor (NGF) and downregulated insulin growth factor (IGF) in UC-hMSCs cultured in bioreactor, while IL4, IL6, IL8, TGFb and VEGF expression levels remained at the similar levels. A culturing of UC-hMSCs using a large-scale automated closed bioreactor expansion system under the GMP conditions does not alter basic "mesenchymal" features and quality of the cells. Our study has been designed to pave a road toward translation of basic research data known about human UC-MSCs for the future clinical testing in patients with neurological and immunocompromised disorders. An industrial manufacturing of UC-hMSCs next will undergo regulatory approval following advanced therapy medicinal products (ATMP) criteria prior to clinical application and approval to be used in patients.

Proteomics Identification and Validation of Desmocollin-1 and Catechol-O-Methyltransferase as Proteins Associated with Breast Cancer Cell Migration and Metastasis.

Biological treatment of many cancers currently targets membrane bound receptors located on a cell surface. To identify novel membrane proteins associated with migration and metastasis of breast cancer cells, a more migrating subpopulation of MDA-MB-231 breast cancer cell line is selected and characterized. A high-resolution quantitative mass spectrometry with SILAC labeling is applied to analyze their surfaceome and it is compared with that of parental MDA-MB-231 cells. Among 824 identified proteins (FDR < 0.01), 128 differentially abundant cell surface proteins with at least one transmembrane domain are found. Of these, i) desmocollin-1 (DSC1) is validated as a protein connected with lymph node status of luminal A breast cancer, tumor grade, and Her-2 status by immunohistochemistry in the set of 96 primary breast tumors, and ii) catechol-O-methyltransferase is successfully verified as a protein associated with lymph node metastasis of triple negative breast cancer as well as with tumor grade by targeted data extraction from the SWATH-MS data of the same set of tissues. The findings indicate importance of both proteins for breast cancer development and metastasis and highlight the potential of biomarker validation strategy via targeted data extraction from SWATH-MS datasets.

Low infiltration of tumor-associated macrophages in high c-Myb-expressing breast tumors.

Tumor-associated macrophages (TAMs) are prominent components of tumor stroma that promotes tumorigenesis. Many soluble factors participate in the deleterious cross-talk between TAMs and transformed cells; however mechanisms how tumors orchestrate their production remain relatively unexplored. c-Myb is a transcription factor recently described as a negative regulator of a specific immune signature involved in breast cancer (BC) metastasis. Here we studied whether c-Myb expression is associated with an increased presence of TAMs in human breast tumors. Tumors with high frequency of c-Myb-positive cells have lower density of CD68-positive macrophages. The negative association is reflected by inverse correlation between MYB and CD68/CD163 markers at the mRNA levels in evaluated cohorts of BC patients from public databases, which was found also within the molecular subtypes. In addition, we identified potential MYB-regulated TAMs recruiting factors that in combination with MYB and CD163 provided a valuable clinical multigene predictor for BC relapse. We propose that identified transcription program running in tumor cells with high MYB expression and preventing macrophage accumulation may open new venues towards TAMs targeting and BC therapy.

High c-Myb Expression Associates with Good Prognosis in Colorectal Carcinoma.

Colorectal cancer (CRC) represents a serious challenge for oncologists due to high incidence and large heterogeneity. Prognostic factors are needed to stratify patients according to risk of disease progression. In this study, we report that high expression of c-Myb protein, determined by immunohistochemistry (IHC), associates with better overall and disease-free survival (OS, DFS) in a cohort of 103 patients. Although MYB has been previously considered to act as oncogene in CRC, our further analysis of datasets deposited in PrognoScan and SurvExpress databases confirmed that high MYB expression largely associates with good prognosis in CRC. As therapies targeting c-Myb have been developed and tested in preclinical studies, we believe that further studies are needed for detailed understanding of c-Myb function in CRC, before the c-Myb-targeted therapy enters clinical trials.

CCL2 Is a Vascular Permeability Factor Inducing CCR2-Dependent Endothelial Retraction during Lung Metastasis.

Increased levels of the chemokine CCL2 in cancer patients are associated with poor prognosis. Experimental evidence suggests that CCL2 correlates with inflammatory monocyte recruitment and induction of vascular activation, but the functionality remains open. Here, we show that endothelial Ccr2 facilitates pulmonary metastasis using an endothelial-specific Ccr2-deficient mouse model (Ccr2ecKO). Similar levels of circulating monocytes and equal leukocyte recruitment to metastatic lesions of Ccr2ecKO and Ccr2fl/fl littermates were observed. The absence of endothelial Ccr2 strongly reduced pulmonary metastasis, while the primary tumor growth was unaffected. Despite a comparable cytokine milieu in Ccr2ecKO and Ccr2fl/fl littermates the absence of vascular permeability induction was observed only in Ccr2ecKO mice. CCL2 stimulation of pulmonary endothelial cells resulted in increased phosphorylation of MLC2, endothelial cell retraction, and vascular leakiness that was blocked by an addition of a CCR2 inhibitor. These data demonstrate that endothelial CCR2 expression is required for tumor cell extravasation and pulmonary metastasis. IMPLICATIONS: The findings provide mechanistic insight into how CCL2-CCR2 signaling in endothelial cells promotes their activation through myosin light chain phosphorylation, resulting in endothelial retraction and enhanced tumor cell migration and metastasis.

Trop-2 plasticity is controlled by epithelial-to-mesenchymal transition.

The cell surface glycoprotein Trop-2 is commonly overexpressed in carcinomas and represents an exceptional antigen for targeted therapy. Here, we provide evidence that surface Trop-2 expression is functionally connected with an epithelial phenotype in breast and prostate cell lines and in patient tumor samples. We further show that Trop-2 expression is suppressed epigenetically or through the action of epithelial-to-mesenchymal transition transcription factors and that deregulation of Trop-2 expression is linked with cancer progression and poor patient prognosis. Moreover, our data suggest that the cancer plasticity-driven intratumoral heterogeneity in Trop-2 expression may significantly contribute to response and resistance to therapies targeting Trop-2-expressing cells.

Monocyte Induction of E-Selectin-Mediated Endothelial Activation Releases VE-Cadherin Junctions to Promote Tumor Cell Extravasation in the Metastasis Cascade.

Tumor cells interact with blood constituents and these interactions promote metastasis. Selectins are vascular receptors facilitating interactions of tumor cells with platelets, leukocytes, and endothelium, but the role of endothelial E-selectin remains unclear. Here we show that E-selectin is a major receptor for monocyte recruitment to tumor cell-activated endothelium. Experimental and spontaneous lung metastasis using murine tumor cells, without E-selectin ligands, were attenuated in E-selectin-deficient mice. Tumor cell-derived CCL2 promoted endothelial activation, resulting in enhanced endothelial E-selectin expression. The recruitment of inflammatory monocytes to metastasizing tumor cells was dependent on the local endothelial activation and the presence of E-selectin. Monocytes promoted transendothelial migration of tumor cells through the induction of E-selectin-dependent endothelial retractions and a subsequent modulation of tight junctions through dephosphorylation of VE-cadherin. Thus, endothelial E-selectin shapes the tumor microenvironment through the recruitment, adhesion, and activation of monocytes that facilitate tumor cell extravasation and thereby metastasis. These findings provide evidence that endothelial E-selectin is a novel factor contributing to endothelial retraction required for efficient lung metastasis. Cancer Res; 76(18); 5302-12. ©2016 AACR.

c-Myb regulates NOX1/p38 to control survival of colorectal carcinoma cells.

The c-Myb transcription factor is important for maintenance of immature cells of many tissues including colon epithelium. Overexpression of c-Myb occurring in colorectal carcinomas (CRC) as well as in other cancers often marks poor prognosis. However, the molecular mechanism explaining how c-Myb contributes to progression of CRC has not been fully elucidated. To address this point, we investigated the way how c-Myb affects sensitivity of CRC cells to anticancer drugs. Using CRC cell lines expressing exogenous c-myb we show that c-Myb protects CRC cells from the cisplatin-, oxaliplatin-, and doxorubicin-induced apoptosis, elevates reactive oxygen species via up-regulation of NOX1, and sustains the pro-survival p38 MAPK pathway. Using pharmacological inhibitors and gene silencing of p38 and NOX1 we found that these proteins are essential for the protective effect of c-Myb and that NOX1 acts upstream of p38 activation. In addition, our result suggests that transcription of NOX1 is directly controlled by c-Myb and these genes are strongly co-expressed in human tumor tissue of CRC patients. The novel c-Myb/NOX1/p38 signaling axis that protects CRC cells from chemotherapy described in this study could provide a new base for design of future therapies of CRC.

Overexpression of c-Myb is associated with suppression of distant metastases in colorectal carcinoma.

The MYB gene codes for the c-Myb transcription factor maintaining proliferation of colon epithelial progenitors, thus controlling colon development and homeostasis. This gene is overexpressed in early phases of colorectal cancer (CRC) tumorigenesis. The aim of this study was to examine the expression of c-Myb in CRC tissue samples both at the messenger RNA (mRNA) and protein levels and to evaluate their associations with clinicopathological characteristics in a group of 108 CRC patients. Statistically significant negative association was found between the frequency of the c-Myb-positive tumor cells assessed by immunohistochemistry and the presence of distant metastases (p < 0.01) but not tumor differentiation, tumor stage, lymph node involvement, vascular invasion, tumor localization, age, and gender of the patients. Although the c-Myb protein level in the tumor tissue correlated with its mRNA level, no significant association between MYB mRNA and any clinicopathological characteristics was observed. We conclude that albeit overexpression of c-Myb is considered as an important factor contributing to early phases of CRC tumorigenesis, it may later have negative effect on tumor cell dissemination as observed recently in breast cancer as well. Further studies are required to explain the role of c-Myb during formation of CRC distant metastases.

Enzymatically active cathepsin D sensitizes breast carcinoma cells to TRAIL.

Cathepsin D (CD), a ubiquitously expressed lysosomal aspartic protease, is upregulated in human breast carcinoma and many other tumor types. CD has been repeatedly reported to act as key mediator of apoptosis induced by various chemotherapeutics. However, there is still controversy over the role of enzymatic/proteolytic versus protein-protein interaction activities of CD in apoptotic signaling. The elucidation of molecular mechanism responsible for the effect of CD in the chemotherapy-induced cell death is crucial for development of an appropriate strategy to target this protease in cancer treatment. Therefore, the objective of this study was to investigate the molecular mechanism behind the CD-mediated regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death. For this purpose, MDA-MB-231 breast carcinoma cells with an increased level of wt CD (CD) or mutant enzymatically inactive CD (ΔCD) were subjected to TRAIL and the frequency of apoptosis was determined. Our results show that CD facilitates the TRAIL-induced apoptosis of MDA-MB-231 breast cancer cells in enzymatic activity-dependent manner. Moreover, the importance of endosomal/lysosomal acidification in this process was documented. Analysis of the potential substrates specifically cleaved by CD during the TRAIL-induced apoptosis confirmed caspase-8 and Bid proteins as the CD targets. Moreover, in search for protein regulators of apoptosis that can be cleaved by CD at physiologically relevant pH, we identified the Bcl-2 protein as a suitable candidate. The modulatory role of CD in cell response to TRAIL was also confirmed in another breast cancer cell line SKBR3. These experiments identified the CD enzymatic activity as a new factor affecting sensitivity of breast cancer cells to TRAIL.

Molecular response of 4T1-induced mouse mammary tumours and healthy tissues to zinc treatment.

Breast cancer patients negative for the nuclear oestrogen receptor α have a particularly poor prognosis. Therefore, the 4T1 cell line (considered as a triple-negative model) was chosen to induce malignancy in mice. The aim of the present study was to assess if zinc ions, provided in excess, may significantly modify the process of mammary oncogenesis. Zn(II) ions were chosen because of their documented antitumour effects. Zn(II) is also known to induce the expression of metallothioneins (MT) and glutathion (GSH). A total dose of zinc sulphate per one gram of mouse weight used in the experiment was 0.15 mg. We studied the expression of MT1, MT2, TP53 and MTF-1 genes and also examined the effect of the tumour on antioxidant capacity. Tumour-free mice had significantly higher expression levels of the studied genes (p<0.003). Significant differences were also revealed in the gene expression between the tissues (p<0.001). The highest expression levels were observed in the liver. As compared to brain, lung and liver, significantly lower concentrations of MT protein were found in the primary tumour; an inverse trend was observed in the concentration of Zinc(II). In non-tumour mice, the amount of hepatic hydrosulphuryl groups significantly increased by the exposure to Zn(II), but the animals with tumour induction showed no similar trend. The primary tumour size of zinc-treated animals was 20% smaller (p=0.002); however, no significant effect on metastasis progression due to the zinc treatment was discovered. In conclusion, Zn(II) itself may mute the growth of primary breast tumours especially at their early stages.

Modulation of induced cytotoxicity of doxorubicin by using apoferritin and liposomal cages.

Doxorubicin is an effective chemotherapeutic drug, however, its toxicity is a significant limitation in therapy. Encapsulation of doxorubicin inside liposomes or ferritin cages decreases cardiotoxicity while maintaining anticancer potency. We synthesized novel apoferritin- and liposome-encapsulated forms of doxorubicin ("Apodox" and "lip-8-dox") and compared its toxicity with doxorubicin and Myocet on prostate cell lines. Three different prostatic cell lines PNT1A, 22Rv1, and LNCaP were chosen. The toxicity of the modified doxorubicin forms was compared to conventional doxorubicin using the MTT assay, real-time cell impedance-based cell growth method (RTCA), and flow cytometry. The efficiency of doxorubicin entrapment was 56% in apoferritin cages and 42% in the liposome carrier. The accuracy of the RTCA system was verified by flow-cytometric analysis of cell viability. The doxorubicin half maximal inhibition concentrations (IC50) were determined as 170.5, 234.0, and 169.0 nM for PNT1A, 22Rv1, and LNCaP, respectively by RTCA. Lip8-dox is less toxic on the non-tumor cell line PNT1A compared to doxorubicin, while still maintaining the toxicity to tumorous cell lines similar to doxorubicin or epirubicin (IC50 = 2076.7 nM for PNT1A vs. 935.3 and 729.0 nM for 22Rv1 and LNCaP). Apodox IC50 was determined as follows: 603.1, 1344.2, and 931.2 nM for PNT1A, 22Rv1, and LNCaP.

Mesenchymal stromal cell labeling by new uncoated superparamagnetic maghemite nanoparticles in comparison with commercial Resovist--an initial in vitro study.

OBJECTIVE: Cell therapies have emerged as a promising approach in medicine. The basis of each therapy is the injection of 1-100×10(6) cells with regenerative potential into some part of the body. Mesenchymal stromal cells (MSCs) are the most used cell type in the cell therapy nowadays, but no gold standard for the labeling of the MSCs for magnetic resonance imaging (MRI) is available yet. This work evaluates our newly synthesized uncoated superparamagnetic maghemite nanoparticles (surface-active maghemite nanoparticles - SAMNs) as an MRI contrast intracellular probe usable in a clinical 1.5 T MRI system. METHODS: MSCs from rat and human donors were isolated, and then incubated at different concentrations (10-200 µg/mL) of SAMN maghemite nanoparticles for 48 hours. Viability, proliferation, and nanoparticle uptake efficiency were tested (using fluorescence microscopy, xCELLigence analysis, atomic absorption spectroscopy, and advanced microscopy techniques). Migration capacity, cluster of differentiation markers, effect of nanoparticles on long-term viability, contrast properties in MRI, and cocultivation of labeled cells with myocytes were also studied. RESULTS: SAMNs do not affect MSC viability if the concentration does not exceed 100 µg ferumoxide/mL, and this concentration does not alter their cell phenotype and long-term proliferation profile. After 48 hours of incubation, MSCs labeled with SAMNs show more than double the amount of iron per cell compared to Resovist-labeled cells, which correlates well with the better contrast properties of the SAMN cell sample in T2-weighted MRI. SAMN-labeled MSCs display strong adherence and excellent elasticity in a beating myocyte culture for a minimum of 7 days. CONCLUSION: Detailed in vitro tests and phantom tests on ex vivo tissue show that the new SAMNs are efficient MRI contrast agent probes with exclusive intracellular uptake and high biological safety.

Cisplatin-resistant prostate cancer model: Differences in antioxidant system, apoptosis and cell cycle.

Differences in the antioxidant system, apoptotic mechanism and in cell cycle between prostatic cell lines could partially elucidate the development of cisplatin resistance. The aim of this study was to identify the most characteristic parameter for a particular cell line and/or a particular cisplatin treatment using a general regression model and to assess whether it is possible to use measured parameters as markers of cisplatin resistance. This study integrates the results of viability, antioxidant, flow cytometric and quantitative PCR assays in order to characterize the resistance of prostate cancer to cisplatin. Cell growth using metabolic- (MTT) and impedance-based assays, the expression of key cell death signaling proteins (p53, Bax and Bcl-2), cell cycle, activity of antioxidant system-related proteins (superoxide dismutase, glutathione peroxidase, glutathione reductase and metallothionein) and free radical scavenging capacity assays [free radicals (FR), ferric reducing antioxidant power (FRAP), ABTS] were analyzed in the cell lines 22Rv1, PC-3 and PNT1A with respect to rising concentrations (0-150 µM) and different length of cisplatin treatment (12-72 h). The non-functional-p53 PC-3 cell line showed decreased BAX (p<0.05) and, in contrast to PNT1A and 22Rv1, no cisplatin-induced effects on cell cycle. All cell lines showed increasing levels of free radical scavenging activity by ABTS, FRAP and FR assays in a time- and dose-dependent manner (r>0.76 at p<0.001 for ABTS, FRAP and FR at p<0.001). PC-3 showed increased (p<0.05) levels of free radical scavenging activity by ABTS and FR methods. These findings, together with significantly elevated MT, decreased p53 and Bax indicate PC-3 to be cisplatin-resistant. The differences in the antioxidant system and apoptotic mechanisms in PC-3 cells may elucidate the development of cisplatin resistance and indicate that this cell line may be further studied as a model of cytostatic resistance.

Nuclear factor of activated T-cells 1 increases sensitivity of v-myb transformed monoblasts to all-trans retinoic acid.

Nuclear factors of activated T-cells (NFATs) are important regulators of the cytokine gene expression in activated T-cells. In the last decade, NFATs have been shown to regulate cell cycle, differentiation and apoptosis in cells of various origins revealing their importance for cell homeostasis. In this study, we investigated the effects of NFAT1 on proliferation and differentiation of v-myb-transformed BM2 monoblasts. In contrast to many other leukemic cell lines, BM2 cells do not respond to retinoic acid. However, once overexpressing NFAT1, they became sensitive to all-trans retinoic acid (ATRA). The ATRA-treated BM2NFAT1 cells differentiated along monocyte/macrophage pathway as evidenced by changes in cell morphology, adherence, phagocytic and non-specific esterase activities, reactive oxygen species production, and vimentin expression. Furthermore, overexpressed NFAT1 either alone or in combination with the ATRA-driven signalling pathway deregulated cyclin A and retinoic acid receptor proteins in BM2 cells. Data presented in this study indicate that the NFAT1 and ATRA signalling pathways synergize in control of proliferation and differentiation of BM2 monoblasts.

Localization of c-MYB in differentiated cells during postnatal molar and alveolar bone development.

The MYB family of transcription activators has been associated with a high proliferation rate and an undifferentiated state of cells in a number of tissues. Recently emerging data suggest that these molecules may also play a role in differentiation. In this study, the pattern of expression of c-MYB was followed during postnatal stages of mouse molar odontogenesis using immunohistochemistry on serial sections. Along with an abundance of the c-MYB protein in proliferating zones, we confirmed the presence of this protein in differentiated ameloblasts, odontoblasts, and osteoblasts. In addition, c-MYB was also found in cementoblasts and alveolar fibroblasts. These findings suggest integration of c-MYB into regulatory networks during hard-tissue differentiation and mineralization.