Chemokine treatment rescues profound T-lineage progenitor homing defect after bone marrow transplant conditioning in mice.

Development of T cells in the thymus requires continuous importation of T-lineage progenitors from the bone marrow via the circulation. Following bone marrow transplant, recovery of a normal peripheral T-cell pool depends on production of naïve T cells in the thymus; however, delivery of progenitors to the thymus limits T-lineage reconstitution. Here, we examine homing of intravenously delivered progenitors to the thymus following irradiation and bone marrow reconstitution. Surprisingly, following host conditioning by irradiation, we find that homing of lymphoid-primed multipotent progenitors and common lymphoid progenitors to the thymus decreases more than 10-fold relative to unirradiated mice. The reduction in thymic homing in irradiated mice is accompanied by a significant reduction in CCL25, an important chemokine ligand for thymic homing. We show that pretreatment of bone marrow progenitors with CCL25 and CCL21 corrects the defect in thymic homing after irradiation and promotes thymic reconstitution. These data suggest new therapeutic approaches to promote T-cell regeneration.

Suppression of renal cell carcinoma growth by inhibition of Notch signaling in vitro and in vivo.

Loss of the tumor suppressor gene von Hippel-Lindau (VHL) plays a key role in the oncogenesis of clear cell renal cell carcinoma (CCRCC). The loss leads to stabilization of the HIF transcription complex, which induces angiogenic and mitogenic pathways essential for tumor formation. Nonetheless, additional oncogenic events have been postulated to be required for the formation of CCRCC tumors. Here, we show that the Notch signaling cascade is constitutively active in human CCRCC cell lines independently of the VHL/HIF pathway. Blocking Notch signaling resulted in attenuation of proliferation and restrained anchorage-independent growth of CCRCC cell lines. Using siRNA targeting the different Notch receptors established that the growth-promoting effects of the Notch signaling pathway were attributable to Notch-1 and that Notch-1 knockdown was accompanied by elevated levels of the negative cell-cycle regulators p21 Cip1 and/or p27 Kip1. Treatment of nude mice with an inhibitor of Notch signaling potently inhibited growth of xenotransplanted CCRCC cells. Moreover, Notch-1 and the Notch ligand Jagged-1 were expressed at significantly higher levels in CCRCC tumors than in normal human renal tissue, and the growth of primary CCRCC cells was attenuated upon inhibition of Notch signaling. These findings indicate that the Notch cascade may represent a novel and therapeutically accessible pathway in CCRCC.

Sequential loss of cell cycle checkpoint control contributes to malignant transformation of murine embryonic fibroblasts induced by 20-methylcholanthrene.

Definitive information about the number and nature of discrete steps of tumorigenesis is enigmatic. To understand the multistep nature of carcinogenesis, an in vitro model of 20-Methylcholanthrene-treated primary fibroblast cells CNCI-PM-20, from 20-day old Swiss mouse embryo was used. Visible neoplastic changes with distinct morphological variations along with specific chromosomal aberrations like Robertsonian metacentrics, double and single-minute chromosomes and aneuploidy were observed from Passage-20 onwards. The cell cycle profile showed gradual increase in G(2)/M population till P-32, followed by evasion of block from P-36 onwards. Gradual increase in expression of C-myc, CyclinD1 and a decrease in expression of P21 was observed from P-20 onwards. CDC25A expression was significantly increased at P-27 and remained more or less constant in subsequent passages. Additionally, an increased P16 and P53 expression were seen at P-20 followed by their significant down-regulation at P-32. An increased level of phosphorylated retinoblastoma (ppRb) was observed from P-27, probably responsible for a compromised G(1)/S checkpoint. The inactivation of p21 and p16 might be due to their promoter hyper-methylation as suggested through de-methylation experiment by 5-aza-deoxycytidine at P-42. G(2)/M checkpoint abrogation was marked by gradual increase in expression of CyclinB1 and Cdc20, and a significant increase of Mad2 at P-20. Interestingly, increased expression of phospho-ATM, ATR and phospho-Chk1 were also seen at P-20 followed by their down-regulation at subsequent passages, indicating a perturbation of DNA damage response pathway at early passages. Our findings therefore dramatize the multiple genetic events that can cooperate to promote tumorigenesis.

Differential alterations in metabolic pattern of the spliceosomal uridylic acid-rich small nuclear RNAs (UsnRNAs) during malignant transformation of 20-methylcholanthrene-induced mouse CNCI-PM-20 embryonic fibroblasts.

Differential alterations of the spliceosomal Uridylic acid rich small nuclear RNAs (UsnRNAs) (U1, U2, U4, U5, and U6) are reported to be associated with cellular proliferation and development, but definitive information is scarce and also elusive. An attempt is made in this study to analyze the metabolic patterns of major spliceosomal UsnRNAs, during tumor development, in an in vitro carcinogenesis model of 20-methylcholanthrene (MCA)-transformed Swiss Mouse Embryonic Fibroblast (MEF), designated as CNCI-PM-20. MEF cells, after treatment with 20-MCA, progressed through a sequence of passages with distinct and heritable changes, finally becoming neoplastic at passage-42 (P42). A differential expression pattern of major UsnRNAs was observed during this process. The abundance of U1 was 20% below control (P1) at passage-20 (P20), followed by a gradual increase up until P42 (approximately 12% above the P1 value). The abundance of U2 was more or less constant during the cellular transformation. U4 showed a trend of increase, with above 30% abundance than control at P20, followed by a significant increase at P36 and P42 (1.5- and 2-fold, respectively, P-value <0.01). U5 also followed an identical pattern, with an increase of 70% compared to control (P-value <0.05) at P42. Interestingly, U6 gradually decreased from P20 onwards up until P42, with 22% at P20 and 67% at P42 (P-value <0.01). An overall significant quantitative alteration in abundance of U4, U5, and U6, observed in our study, contributes to the understanding of the fact that, the metabolism of major spliceosomal UsnRNAs is differentially regulated during the process of neoplastic transformation.

Black tea polyphenols restrict benzopyrene-induced mouse lung cancer progression through inhibition of Cox-2 and induction of caspase-3 expression.

Lung cancer is one of the leading causes of cancer related death in most developed and many developing countries of the world. Due to lack of validated screening methods and poor prognosis, treatment of lung cancer has not improved significantly over the last two decades. Therefore the risk of the disease needs to be minimized by preventive measures. One approach for lung cancer prevention envisages reversal or restriction of precancerous lesions by chemopreventive intervention. It demands a deeper understanding of the pathogenesis of the disease and identification of the ideal point of intervention. In the present investigation, tea components, epigallocatechin gallate (EGCG) and theaflavins (TF) were assessed for their chemopreventive potential when administered in the post initiation phase of lung carcinogenesis in an experimental mouse model. Histopathological changes in lungs of mice administered benzo(a)pyrene (BP) were followed serially and correlated with the expression of Cox-2, caspase-3 and caspase-7, which play key roles in histopathogenesis of neoplasia. The observations strongly indicate that both EGCG and TF can influence the expression of these genes to modulate the process of carcinogenesis, resulting in delayed onset and lowered incidence of pre-invasive lung lesions.

Intrathymic Injection.

Intrathymic injection is used in several T cell-associated immunological studies to deliver cells or other substances directly into the thymus. Here, we describe the intrathymic injection procedure involving surgical incision of the mouse with or without a thoracotomy. Though this procedure can result in poor recovery, postsurgical complications, and distress to the animal, it is actually a simple procedure that can be carried out relatively easily and quickly with experience.

Black tea extract can modulate protein expression of H-ras, c-Myc, p53, and Bcl-2 genes during pulmonary hyperplasia, dysplasia, and carcinoma in situ.

Lung cancer has emerged as one of the leading causes of cancer death in most developed and many developing countries of the world. In the absence of effective screening and early detection methods of lung cancer and overall poor prognosis, the 5-year survival following treatment has not improved significantly over the last two decades. It is hoped that the risk of the disease can be minimized by preventive measures. One aspect of lung cancer prevention emphasizes the cessation of tobacco smoking, and another strategy envisages reversal or restriction of the process of lung carcinogenesis by chemopreventive intervention. The latter strategy, however, demands a deeper understanding of the pathogenesis of the disease and the identification of the ideal point of intervention. In the present investigation, we assessed the role of the antioxidant tea components theaflavins (TF) and epigallocatechin gallate (EGCG) for their chemopreventive potential and molecular mechanism of action when administered at the post-initiation phase of lung carcinogenesis in an experimental mouse model. We serially examined the histopathological changes in the lung of mice administered benzo(a)pyrene and correlated them with the frequency of proliferative and apoptotic cells in situ as well as with the expression of H-ras, c-Myc, p53, and Bcl-2 genes, which play key roles in the histopathogenesis of neoplasia. Our findings indicate that both TF and EGCG can influence gene expression to modulate the process of carcinogenesis through the regulation of apoptosis. This results in a lowered incidence and delayed onset of preinvasive lung lesions.

Histone H3 Lysine 27 demethylases Jmjd3 and Utx are required for T-cell differentiation.

Although histone H3 lysine 27 trimethylation (H3K27Me3) is associated with gene silencing, whether H3K27Me3 demethylation affects transcription and cell differentiation in vivo has remained elusive. To investigate this, we conditionally inactivated the two H3K27Me3 demethylases, Jmjd3 and Utx, in non-dividing intrathymic CD4(+) T-cell precursors. Here we show that both enzymes redundantly promote H3K27Me3 removal at, and expression of, a specific subset of genes involved in terminal thymocyte differentiation, especially S1pr1, encoding a sphingosine-phosphate receptor required for thymocyte egress. Thymocyte expression of S1pr1 was not rescued in Jmjd3- and Utx-deficient male mice, which carry the catalytically inactive Utx homolog Uty, supporting the conclusion that it requires H3K27Me3 demethylase activity. These findings demonstrate that Jmjd3 and Utx are required for T-cell development, and point to a requirement for their H3K27Me3 demethylase activity in cell differentiation.

The notch and TGF-ß signaling pathways contribute to the aggressiveness of clear cell renal cell carcinoma.

BACKGROUND: Despite recent progress, therapy for metastatic clear cell renal cell carcinoma (CCRCC) is still inadequate. Dysregulated Notch signaling in CCRCC contributes to tumor growth, but the full spectrum of downstream processes regulated by Notch in this tumor form is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We show that inhibition of endogenous Notch signaling modulates TGF-ß dependent gene regulation in CCRCC cells. Analysis of gene expression data representing 176 CCRCCs showed that elevated TGF-ß pathway activity correlated significantly with shortened disease specific survival (log-rank test, p = 0.006) and patients with metastatic disease showed a significantly elevated TGF-ß signaling activity (two-sided Student's t-test, p = 0.044). Inhibition of Notch signaling led to attenuation of both basal and TGF-ß1 induced TGF-ß signaling in CCRCC cells, including an extensive set of genes known to be involved in migration and invasion. Functional analyses revealed that Notch inhibition decreased the migratory and invasive capacity of CCRCC cells. CONCLUSION: An extensive cross-talk between the Notch and TGF-ß signaling cascades is present in CCRCC and the functional properties of these two pathways are associated with the aggressiveness of this disease.

Tea polyphenols can restrict benzo[a]pyrene-induced lung carcinogenesis by altered expression of p53-associated genes and H-ras, c-myc and cyclin D1.

The modulatory influence of tea polyphenols (epigallocatechin gallate, epicatechin gallate and theaflavin) on benzo[a]pyrene (B[a]P)-induced lung carcinogenesis in mice was analyzed using histopathological and molecular parameters. Progression of lung lesions was restricted at the hyperplastic stage by tea polyphenols. A significant reduction in cellular proliferative index and an increase in apoptotic index were noted in the restricted lung lesions. High expression of H-ras, c-myc, cyclin D1 and p53 genes was seen at the inflammatory stage (9th week) and in subsequent premalignant lesions, but down-regulation of H-ras at the hyperplastic stage (17th week). Expression of bcl-2 was high in hyperplastic lesions, whereas the expression of mdm2 and bcl-xl increased only at the moderately dysplastic stage (36th week). The tea polyphenols inhibited inflammatory response in the lung lesions on the 9th week, when decreased expression of H-ras and c-myc and increased expression of bax were noted. Prolonged treatment (>9th week) with tea polyphenols resulted in changes in the expression of some additional genes, such as reduced expression of cyclin D1 (from the 17th week), bcl-2 (from the 26th week; mild dysplasia) and p21 (on the 36th week), and high expression of p53 (from the 17th week) and p27 (on the 36th week). These observations indicate that the tea polyphenols can restrict B[a]P-induced lung carcinogenesis by differential modulation of the expression of p53 and its associated genes such as bax, bcl-2, mdm2, p21 and p27, along with H-ras, c-myc and cyclin D1, at different time points.

Epigallocatechin gallate induced apoptosis in Sarcoma180 cells in vivo: mediated by p53 pathway and inhibition in U1B, U4-U6 UsnRNAs expression.

The aim of this study was to understand the mode of action of tea polyphenol epigallocatechin gallate (EGCG) in vivo. Swiss albino mice were treated i.p. with EGCG at two different doses i.e. 12-mg/kg body weight and 15-mg/kg body weight, for 7 days prior to inoculation of Sarcoma180 (S180) cells and continued for another 7 days. The growth of the S180, harvested 7 days after inoculation, was significantly reduced due to treatment with EGCG. The flowcytometric analysis of S180 cells, showed significant increase in apoptosis and reduction in the number of cells in G2/M phase of cell cycle due to treatment with EGCG. The induction of apoptosis has also been confirmed by the TUNEL and DNA fragmentation assays. Both RT-PCR and Western blot analysis showed significant up-regulation of p53 and bax, and down-regulation of bcl-2 and c-myc due to EGCG treatment. No changes in the expression pattern of p21, p27, bcl-xl, mdm2 and cyclin D1 were seen. Interestingly, there was significant down-regulation of spliceosomal uridylic acid rich small nuclear RNAs (UsnRNAs) U1B and U4-U6 due to EGCG treatment. This indicates that these UsnRNAs may be involved in the apoptosis process. Taken together, our study suggests that in vivo EGCG could induce apoptosis in S180 cells through alteration in G2/M phase of the cell cycle by up-regulation of p53, bax and down-regulation of c-myc, bcl-2 and U1B, U4-U6 UsnRNAs.

Differential alterations in metabolic pattern of the spliceosomal UsnRNAs during pre-malignant lung lesions induced by benzo(a)pyrene: modulation by tea polyphenols.

The differential alterations of the spliceosomal UsnRNAs (U1, U2, U4, U5, and U6) were reported to be associated with cellular proliferation and development. The attempt was made in this study to analyze the metabolic pattern of the spliceosomal UsnRNAs during the development of pre-malignant lung lesions induced in experimental mice model system by benzo(a)pyrene (BP) and also to see how tea polyphenols, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), modulate the metabolism of these UsnRNAs during the lung carcinogenesis. No significant changes in the level of the UsnRNAs were seen in the inflammatory lung lesions at 9th week due to treatment of BP. However, there was significant increase in the level of U1 ( approximately 2.5 fold) and U5 ( approximately 47%) in the hyperplastic lung lesions at 17th week. But in the mild dysplastic lung lesions at 26th week, the level of UsnRNAs did not change significantly. Whereas, in the dysplastic lung lesions at 36th week there was significant increase in the level of the U2 ( approximately 2 fold), U4 ( approximately 2.5 fold) and U5 ( approximately 2 fold). Due to the EGCG and ECG treatment the lung lesions at 9th week appeared normal and in the 17th, 26th, and 36th week it appeared as hyperplasia. The level of the UsnRNAs was significantly low in the lung lesions at 9th week (only U2 and U4 by EGCG), at 17th week (only U1 by EGCG/ECG), at 26th week (U1 by ECG; U2, U4 and U5 by EGCG/ECG) and at 36th week (U1 by ECG, U2 and U4 by EGCG/ECG). Whereas, there was significant increase in the level of U5 (by EGCG/ECG) and U6 (by EGCG only) in the lung lesions at 36th and 26th week respectively. This indicates that the metabolism of the spliceosomal UsnRNAs differentially altered during the development of pre-malignant lung lesions by BP as well as during the modulation of the lung lesions by the tea polyphenols.