Biomedical subjects
A Bhushan
Publications and source records attributed to A Bhushan.
Sustained beta cell apoptosis in patients with long-standing type 1 diabetes: indirect evidence for islet regeneration?
AIMS/HYPOTHESIS: Type 1 diabetes is widely held to result from an irreversible loss of insulin-secreting beta cells. However, insulin secretion is detectable in some people with long-standing type 1 diabetes, indicating either a small population of surviving beta cells or continued renewal of beta cells subject to ongoing autoimmune destruction. The aim of the present study was to evaluate these possibilities. MATERIALS AND METHODS: Pancreatic sections from 42 individuals with type 1 diabetes and 14 non-diabetic individuals were evaluated for the presence of beta cells, beta cell apoptosis and replication, T lymphocytes and macrophages. The presence and extent of periductal fibrosis was also quantified. RESULTS: Beta cells were identified in 88% of individuals with type 1 diabetes. The number of beta cells was unrelated to duration of disease (range 4-67 years) or age at death (range 14-77 years), but was higher (p<0.05) in individuals with lower mean blood glucose. Beta cell apoptosis was twice as frequent in type 1 diabetes as in control subjects (p<0.001), but beta cell replication was rare in both groups. The increased beta cell apoptosis in type 1 diabetes was accompanied by both increased macrophages and T lymphocytes and a marked increase in periductal fibrosis (p<0.001), implying chronic inflammation over many years, consistent with an ongoing supply of beta cells. CONCLUSIONS/INTERPRETATION: Most people with long-standing type 1 diabetes have beta cells that continue to be destroyed. The mechanisms underlying increased beta cell death may involve both ongoing autoimmunity and glucose toxicity. The presence of beta cells despite ongoing apoptosis implies, by definition, that concomitant new beta cell formation must be occurring, even after long-standing type 1 diabetes. We conclude that type 1 diabetes may be reversed by targeted inhibition of beta cell destruction.
Autocrine production of interleukin 6 causes multidrug resistance in breast cancer cells.
It has been shown that serum levels of interleukin (IL)-6 are elevated in patients with various types of cancer. However, the exact source of IL-6 in these patients and its role in tumor progression remain unclear. Here we demonstrate that the autocrine production of IL-6 by tumor cells promotes resistance of the cells to chemotherapy, a novel function of IL-6 in cancer biology. Breast cancer cells that are sensitive to drug treatment do not express IL-6, whereas high levels of IL-6 are produced by multidrug-resistant breast cancer cells. Expression of the IL-6 gene in drug-sensitive breast cancer cells increases their resistance to drug treatment by activating the CCAAT enhancer-binding protein family of transcription factors and inducing mdr1 gene expression. Thus, the autocrine production of IL-6 by tumor cells is an important factor in determining the susceptibility or resistance of these cells to drug treatment. Because tumors from some breast cancer patients contain IL-6-producing cells, it is possible that IL-6 could potentially be used as a prognostic factor for chemotherapy resistance.
CREB/ATF proteins enhance the basal and CD154- and IL-4-induced transcriptional activity of the human Igamma1 proximal promoter.
To understand the underlying basis for the strong IL-4- and CD154-mediated Igamma1 promoter activity in Ramos 2G6 B cells, we carried out transient transfection assays with luciferase-based constructs containing approximately 2.2 kb and 500 bp of the human Igamma1 proximal promoter region. As a comparison, the corresponding regions of the human Igamma3 promoter were tested under identical conditions. We found that both Igamma1 and Igamma3 promoter constructs were activated upon transfection into Ramos B cells and that activity was significantly up-regulated by CD154 and IL-4 signals. However, the Igamma1 promoter was measurably stronger than the Igamma3 promoter with respect to both basal and induced responses. Sequence comparison revealed a divergent 36-bp region containing multiple putative transcription factor binding sites in the Igamma1 but not the Igamma3 promoter. A mutational "swap" of this sequence resulted in a marked decrease and increase in Igamma1 and Igamma3 basal and induced promoter activity, respectively. Gel retardation assays with Igamma1-specific probes revealed CREB-containing complexes that were not observed with the corresponding Igamma3 probes. Mutation of a single nucleotide in overlapping CREB sites in the Igamma1 sequence resulted in a significant decrease in basal activity with a corresponding reduction in the level of IL-4- and CD154-mediated transcription.
Fgf10 is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis.
The importance of mesenchymal-epithelial interactions for the proper development of the pancreas has been acknowledged since the early 1960s, even though the molecule(s) mediating this process have remained unknown. We demonstrate here that Fgf10, a member of the fibroblast growth factor family (FGFs), plays an essential role in this process. We show that Fgf10 is expressed in the mesenchyme directly adjacent to the early dorsal and ventral pancreatic epithelial buds. In Fgf10(-/-) mouse embryos, the evagination of the epithelium and the initial formation of the dorsal and ventral buds appear normal. However, the subsequent growth, differentiation and branching morphogenesis of the pancreatic epithelium are arrested; this is primarily due to a dramatic reduction in the proliferation of the epithelial progenitor cells marked by the production of the homeobox protein PDX1. Furthermore, FGF10 restores the population of PDX1-positive cells in organ cultures derived from Fgf10(-/-) embryos. These results indicate that Fgf10 signalling is required for the normal development of the pancreas and should prove useful in devising methods to expand pancreatic progenitor cells.
CD40:CD40L interactions in X-linked and non-X-linked hyper-IgM syndromes.
Hyper-IgM (HIM) syndrome is a rare immunodeficiency characterized by low or absent IgG, IgA, and IgE with normal or elevated levels of IgM. This disorder can be acquired or familial with either X-linked or autosomal patterns of inheritance. The X-linked form of the disease is a consequence of mutations in the CD40 ligand (CD40L) gene that encodes a protein expressed primarily on activated CD4+ T cells. The cognate interaction between CD40L on T cells and CD40 on antigen-stimulated B cells, macrophage, and dendritic cells is critical for the development of a comprehensive immune response. The non-X-linked form of HIM syndrome is heterogeneous and appears in some cases to be a consequence of mutations in the AlD gene which encodes a B cell specific protein required for class switch recombination, somatic mutation, and germinal center formation. However, mutations in other unidentified genes are clearly the basis of the disease in a subset of patients. In this article, we review the essential features of the X-linked and non-X-linked forms of HIM syndrome and discuss the critical role the CD40:CD40L receptor-ligand pair plays in the pathogenesis of these immune deficiencies.
A transcriptional defect underlies B lymphocyte dysfunction in a patient diagnosed with non-X-linked hyper-IgM syndrome.
To establish the underlying cause of hyper-IgM syndrome in one female patient, B cell function was examined in response to CD40- and IL-4-mediated pathways. When CD40-induced functional responses were measured in unfractionated B cells, CD80 up-regulation, de novo Cmu-Cgamma recombination, and Igamma transcription were all found to be relatively unaffected. However, CD40- and IL-4-mediated CD23 up-regulation and VDJ-Cgamma transcription were clearly diminished compared to control cells. IL-4-induced CD23 expression was measurably reduced in the CD20- population as well. These results suggested that the patient's defect is positioned downstream of CD40 contact and affects both CD40- and IL-4 signal transduction pathways. Further analysis of B cell function in CD19+ B cells revealed a clear B cell defect with respect to Igamma and mature VDJ-Cgamma transcription and IgG expression. However, under the same conditions Iepsilon transcription was relatively normal. Partial restoration of B cell function occurred if PBMC or CD19+ B cells were cultured in vitro in the presence of CD154 plus IL-4. Because addition of IL-4 to cocultures containing activated T cells failed to induce B cells to undergo differentiation, the ability of the patient's B cells to acquire a responsive phenotype correlated with receiving a sustained signal through CD40. These findings support a model in which the patient expresses an intrinsic defect that is manifested in the failure of specific genes to become transcriptionally active in response to either CD154 or IL-4 and results in a functionally unresponsive B cell phenotype.
A polymorphic CD40 ligand (CD154) molecule mediates CD40-dependent signalling but interferes with the ability of soluble CD40 to functionally block CD154:CD40 interactions.
We report the characterization of a naturally occurring polymorphism in CD40 ligand (CD40L, CD154) expressed by activated T cells from a young female patient. This polymorphism encodes a nonconservative Gly --> Arg substitution in amino acid 219 in the extracellular, CD40 binding domain of the molecule. Studies carried out with 293 epithelial cells ectopically expressing the polymorphic protein (CD154/G219R) revealed reduced levels of binding to different anti-CD154 monoclonal antibodies (mAb) and CD40-immunoglobulin (CD40-Ig). However, recognition of the polymorphic and wild-type CD154 molecules by a polyclonal antiserum was comparable, suggesting that the polymorphism affects the ability of the protein to interact with CD40 but does not significantly alter its surface expression. To determine if reduced cross-linking of CD40 mediated decreased functional effects, three CD40-dependent properties were measured. We found that pathways leading to the induction of surface CD23, CD80, and Igamma transcription were activated in response to CD154/G219R signalling. However, the decrease in affinity for CD40 by the mutated CD154 affected the ability of CD40-Ig to efficiently interfere with the binding and effectively block induced CD80 expression. In contrast, we found that the 5c8 mAb, which recognized the polymorphic molecule to a similar extent as wild-type CD154, effectively blocked the interaction between CD154/G219R and CD40 as measured by CD80 expression. These findings suggest that naturally occurring polymorphisms in the CD154 molecule may affect the ability of CD40-mediated functions to be blocked by soluble CD40 or anti-CD154 mAb in the therapeutic treatment of disease and graft rejection.
Impaired membrane transport in methotrexate-resistant CCRF-CEM cells involves early translation termination and increased turnover of a mutant reduced folate carrier.
The basis for impaired reduced folate carrier (RFC) activity in methotrexate-resistant CCRF-CEM (CEM/Mtx-1) cells was examined. Parental and CEM/Mtx-1 cells expressed identical levels of the 3. 1-kilobase RFC transcript. A approximately 85-kDa RFC protein was detected in parental cells by photoaffinity labeling and on Western blots with RFC-specific antiserum. In CEM/Mtx-1 cells, RFC protein was undetectable. By reverse transcriptase-polymerase chain reaction and sequence analysis, G to A point mutations were identified in CEM/Mtx-1 transcripts at positions 130 (P1; changes glycine 44 --> arginine) and 380 (P2; changes serine 127 --> asparagine). A 4-base pair (CATG) insertion detected at position 191 (in 19-30% of cDNA clones) resulted in a frameshift and early translation termination. Wild-type RFC was also detected (0-9% of clones). Wild-type RFC and double-mutated RFC (RFCP1+P2) cDNAs were transfected into transport-impaired K562 and Chinese hamster ovary cells. Although RFC transcripts paralleled wild-type protein, for the RFCP1+P2 transfectants, disproportionately low RFCP1+P2 protein was detected. This reflected an increased turnover of RFCP1+P2 over wild-type RFC. RFCP1+P2 did not restore methotrexate transport; however, uptake was partially restored by constructs with single mutations at the P1 or P2 loci. Cumulatively, our results show that loss of transport function in CEM/Mtx-1 cells results from complete loss of RFC protein due to early translation termination and increased turnover of a mutant RFC protein.
RLIM inhibits functional activity of LIM homeodomain transcription factors via recruitment of the histone deacetylase complex.
LIM domains are required for both inhibitory effects on LIM homeodomain transcription factors and synergistic transcriptional activation events. The inhibitory actions of the LIM domain can often be overcome by the LIM co-regulator known as CLIM2, LDB1 and NLI (referred to hereafter as CLIM2; refs 2-4). The association of the CLIM cofactors with LIM domains does not, however, improve the DNA-binding ability of LIM homeodomain proteins, suggesting the action of a LIM-associated inhibitor factor. Here we present evidence that LIM domains are capable of binding a novel RING-H2 zinc-finger protein, Rlim (for RING finger LIM domain-binding protein), which acts as a negative co-regulator via the recruitment of the Sin3A/histone deacetylase corepressor complex. A corepressor function of RLIM is also suggested by in vivo studies of chick wing development. Overexpression of the gene Rnf12, encoding Rlim, results in phenotypes similar to those observed after inhibition of the LIM homeodomain factor LHX2, which is required for the formation of distal structures along the proximodistal axis, or by overexpression of dominant-negative CLIM1. We conclude that Rlim is a novel corepressor that recruits histone deacetylase-containing complexes to the LIM domain.
Inhibition of phospholipase C-gamma1 activation blocks glioma cell motility and invasion of fetal rat brain aggregates.
OBJECTIVE: Phospholipase C (PLC)-gamma is a cytosolic enzyme activated by several growth factor (GF) receptors (epidermal GF receptor [EGFR], platelet-derived GF receptor, and insulin-like GF 1 receptor), and its activation is associated with increased cell motility (but not cell proliferation) in nonglioma cell lines. Because up-regulated activation of EGFR has been consistently linked to poor patient survival in patients with glioblastoma multiforme (GBM) and because inhibition of EGFR activation by tyrosine kinase inhibitors prevents glioma infiltration in vitro, we hypothesized that inhibition of PLC-gamma activation would inhibit glioma cell invasiveness. METHODS: Our experimental model assesses tumor spheroid invasion of fetal rat brain spheroids by confocal microscopy. We treated U87 GBM spheroids, and those derived from a single patient, with the PLC inhibitor U73122. We also transfected rat C6 glioma cells with the PLCz complementary deoxyribonucleic acid coding for a dominant negative PLC-gamma1 src-homology-2/src-homology-3 peptide fragment, which blocks binding and activation of PLC-gamma1 by GF receptors. Two clones (C6F and C6E) were grown into spheroids and were tested for invasiveness in the spheroid model and for responsiveness to GFs in a standard in vitro motility assay. RESULTS: The infiltration rate of the patient GBM cell line overexpressing wild-type EGFR was reduced by 2 micromol/L U73122 from a slope (percent invasion/h) of 0.74+/-0.08 (with the inactive congener U73343) to 0.04+/-0.053 (P = 8 x 10(-7) by two-tailed t test, 92% reduction); the integral rate, another measure of invasion, was reduced from 49.7+/-13 percent-hours per hour to 13.6+/-12 (P = 0.002, 72% reduction). The U87 spheroid invasion rate was reduced by 0.5 micromol/L U73122 from 46.7+/-8.5 percent-hours per hour to 11.2+/-4.6 (P = 3 x 10(-5)); the slope decreased from 1.7+/-0.41 percent per hour to 0.35+/-0.14 (P = 0.0001). The C6F and C6E clones demonstrated attachment to and "surrounding" of the fetal rat brain aggregate but no true invasion by confocal or light microscopy. PLCz blocked the motility response to epidermal GF, platelet-derived GF, and insulin-like GF. There was a significant decrease in PLC-gamma1-associated tyrosine phosphorylation. CONCLUSION: These results support a key role for PLC-gamma activation as a common postreceptor pathway for GF-induced tumor infiltration and further identify PLC-gamma1 as a possible target for anti-invasive therapy for GBMs.
Collateral methotrexate resistance in cisplatin-selected murine leukemia cells.
Resistance to anticancer drugs is a major cause of failure of many therapeutic protocols. A variety of mechanisms have been proposed to explain this phenomenon. The exact mechanism depends upon the drug of interest as well as the tumor type treated. While studying a cell line selected for its resistance to cisplatin we noted that the cells expressed a > 25,000-fold collateral resistance to methotrexate. Given the magnitude of this resistance we elected to investigate this intriguing collateral resistance. From a series of investigations we have identified an alteration in a membrane protein of the resistant cell as compared to the sensitive cells that could be the primary mechanism of resistance. Our studies reviewed here indicate decreased tyrosine phosphorylation of a protein (molecular mass = 66) in the resistant cells, which results in little or no transfer of methotrexate from the medium into the cell. Since this is a relatively novel function for tyrosine phosphorylation, this information may provide insight into possible pharmacological approaches to modify therapeutic regimens by analyzing the status of this protein in tumor samples for a better survival of the cancer patients.
Smad7 inhibits mesoderm formation and promotes neural cell fate in Xenopus embryos.
We report the isolation and characterization of a new inhibitory Smad in Xenopus, which we have designated as Xenopus Smad7. Smad7 is present at fairly constant levels throughout early development and at blastula stages enriched in the ventral side of the animal hemisphere. The induction of mesoderm by TGF-beta-like signals is mediated by receptor ALK-4 and we show that Smad7 blocks signaling of ALK-4 in a graded fashion: lower levels of Smad7 block activation of dorsal mesoderm genes and higher levels block all mesoderm genes expression. Smad7 is able to directly activate neural markers in explants in the absence of mesoderm or endoderm. This neural-inducing activity of Smad7 may be due to inhibition of BMP-4 signaling because Smad7 can also block BMP-4-mediated mesoderm induction. Thus, Smad7 acts as a potent inhibitor of mesoderm formation and also activates the default neural induction pathway.
Drug resistance results in alterations in expression of immune recognition molecules and failure to express Fas (CD95).
It is demonstrated that methotrexate/cisplatin-sensitive L1210 cells express low levels of major histocompatibility complex (MHC) class II relative to the high levels expressed on methotrexate (MTX)/cisplatin-resistant L1210/DDP cells. L1210 cells express cell-surface Fas, while the L1210/DDP cells express no cell-surface Fas. Expression of costimulatory molecules B7-1/B7-2 and Fas is increased on L1210 cells, but not L1210/DDP, in the presence of methotrexate or trimetrexate (TMTX). Therefore, a component of the mechanism of action of some anti-cancer agents may be to facilitate immune recognition and T cell-directed, Fas-induced cell death. Loss of cell-surface Fas expression and failure of Fas (CD95)-dependent apoptotic death has been observed when cells develop drug resistance. The defect in apoptosis can be overcome by anti-cancer agents or experimental manipulation that induce Fas expression on the drug-resistant cells.
Smad8 mediates the signaling of the ALK-2 [corrected] receptor serine kinase.
Smad proteins are critical intracellular mediators of signaling by growth and differentiation factors of the transforming growth factor beta superfamily. We have isolated a member of the Smad family, Smad8, from a rat brain cDNA library and biochemically and functionally characterized its ability to transduce signals from serine kinase receptors. In Xenopus embryo, Smad8 is able to transcriptionally activate a subset of mesoderm target genes similar to those induced by the receptor serine kinase, activin receptor-like kinase (ALK)-2. Smad8 can be specifically phosphorylated by a constitutively active ALK-2 but not the related receptor serine kinase, ALK-4. In response to signaling from ALK-2, Smad8 associates with a common regulatory molecule, Smad4, and this association leads to a synergistic effect on gene transcription. Furthermore, Smad8 is able to rescue the expression of mesoderm genes blocked by truncated ALK-2 in the embryo. These results indicate that Smad8 can function as a downstream signaling mediator of ALK-2.
Human K562 transfectants expressing high levels of reduced folate carrier but exhibiting low transport activity.
A human reduced folate carrier (hRFC) cDNA was transfected into transport-deficient K562 cells to circumvent complications that may result from carrier expression in a heterologous mammalian species. Relative to wild-type cells, hRFC transcript levels were increased 11- and 19-fold, respectively, in the K43-6 and K43-1 transfectants. Although photoaffinity labeling of hRFC protein revealed similar increases of 15- and 19-fold, respectively, only a 2-fold enhancement in methotrexate (Mtx) transport was observed. This suggests that only a small portion of the cDNA-encoded hRFC protein is actively engaged in membrane transport. Kinetic analysis of [3H]Mtx transport indicated that K43-6 cells exhibited a similar affinity (Kt) but an increased Vmax (1.7-fold) when compared with K562 cells. The restored transport was similar to that of wild-type cells in its capacity to be trans-stimulated by intracellular folates and in its sensitivity to competitive transport inhibitors (1843U89, bromosulfophthalein, folic acid, leucovorin, and ZD1694) and to irreversible inhibition by N-hydroxysuccinimide-methotrexate. Further, deglycosylated photoaffinity-labeled hRFC protein in both K562 and K43-6 cells migrated at approximately 65-70 kDa on SDS-gels, consistent with the molecular mass from the predicted amino acid sequence. These data further establish that the expression of hRFC, alone, is sufficient to confer transport properties typical of the "classical" hRFC. However, the discrepancy between the stoichiometry of carrier expression and transport activity implies that membrane translocation of bound substrate may be regulated by additional undefined mechanisms.
Inhibition of epidermal growth factor receptor-associated tyrosine kinase blocks glioblastoma invasion of the brain.
OBJECTIVE: Glioblastoma multiforme is a malignant primary brain tumor associated with short patient survival despite aggressive treatment, in part because of its propensity to aggressively infiltrate into brain tissue. Glioblastoma multiforme is also unique because it is the only nonepithelial human tumor for which excessive activation of epidermal growth factor receptor (EGFR) has been consistently linked to tumor growth and patient survival, and EGFR activation promotes glioblastoma multiforme infiltration in vitro. METHODS: Cocultures of human glioblastoma spheroids (derived from three separate patients) and fetal rat brain aggregates were examined for infiltration using confocal microscopy, in the presence of 0 to 100 mumol/L genistein, a tyrosine kinase (TK) inhibitor, and 3 mumol/L tyrphostin A25, a specific EGFR-TK inhibitor. RESULTS: Infiltration (not attachment) was completely inhibited by genistein at 10 mumol/L, the IC20 for monolayer growth inhibition in two cell lines. Tyrphostin A25 at 3 mumol/L (the IC20 for monolayers) reduced invasion in a third cell line from 38.8 +/- 6.1% invasion-hour per hour (n = 5) to 2.9 +/- 1.2% invasion-hour per hour (n = 6) (P = 0.0002, two-tailed t test, 93% inhibition), and from 0.54 +/- 0.065% per hour (slope) to 0.028 +/- 0.018% per hour (P = 0.00001, 95% inhibition). Maximal percent invasion was reduced from 100 +/- 0 to 7.4 +/- 5.6% of the fetal rat brain aggregate. No change was detected in EGFR-associated tyrosine phosphorylation at those doses in monolayers by 32P immunolabeling, consistent with the known effects of low concentrations of TK inhibitors. An increase in expression of wild-type and truncated EGFR was demonstrated by Western blotting. Invasion was equally well inhibited by a monoclonal antibody to the high-affinity ligand binding domain of EGFR and not by antibody to an inactive domain. CONCLUSION: Our observations support the role of EGFR activation as a determinant by which glioblastoma invades normal brain tissue, and we show that invasion can be effectively inhibited at much lower concentrations of TK inhibitors than are necessary for growth suppression.
Expression of c-fos precedes MDR3 in vincristine and adriamycin selected multidrug resistant murine erythroleukemia cells.
Expression of murine P-glycoprotein (P-gp), encoded by mdrl or mdri3, confers a multidrug resistance phenotype. Higher expression of c-fos and c-jun has also been demonstrated in multidrug resistant human and murine cells. We detected increased expression of c-fos early in the derivation of two series of murine erythroleukemia sublines selected for resistance to vincristine or adriamycin which eventually overexpress mdr3. We speculate that early expression of c-fos prepares cells for overexpression of other genes, such as mdr3, that contribute to the multidrug resistance phenotype.