Expression of multiple myeloma associated markers in bone marrow spicules using a novel immunohistochemical technique.

Immunohistochemistry (IHC) is an important tool used for diagnosis and prognosis of several hematological malignancies, and it frequently is used for quantitative and qualitative analysis of expression of different protein biomarkers in tissue sections. To understand the histopathological alterations in multiple myeloma (MM), IHC analysis of bone marrow (BM) biopsy is commonly used. Owing to the harsh decalcification process generally used for processing of bone marrow biopsies, however, protein epitopes occasionally are rendered unsuitable for IHC detection. We have developed a novel technique for processing BM spicule samples into a fibrin clot matrix that allows IHC detection of MM protein markers. This method does not require decalcification and results in a consistent, reliable assay. Using paired BM spicule-clot and BM core biopsies from patients diagnosed with multiple myeloma, we studied six MM related antibodies including kappa and lambda immunoglobulin light chains, CD56, CD138, CYR61 and DKK1.

Identification and mapping QTL for high-temperature adult-plant resistance to stripe rust in winter wheat (Triticum aestivum L.) cultivar 'Stephens'.

High-temperature adult-plant (HTAP) resistance from the winter wheat (Triticum aestivum) cultivar 'Stephens' has protected wheat crops from stripe rust caused by Puccinia striiformis f. sp. tritici for 30 years. The objectives of this study were to identify quantitative trait loci (QTL) for HTAP resistance in Stephens through genetic linkage analysis and identify DNA markers linked to the QTL for use in marker-assisted breeding. Mapping populations consisted of 101 recombinant inbred lines (RILs) through single-seed descent from 'Stephens' (resistant) x 'Michigan Amber' (susceptible). F(5), F(6) and F(7) RILs were evaluated for stripe rust resistance at Pullman, WA in 1996, 1997 and 1998, respectively, whereas F(8) RILs were evaluated at Mt Vernon, WA, USA in 2005. The 101 F(8) RILs were evaluated with 250 resistance gene analog polymorphism (RGAP), 245 simple sequence repeat (SSR) and 1 sequence tagged site (STS) markers for genetic linkage map construction. Two QTL, which explained 48-61% of the total phenotypic variation of the HTAP resistance in Stephens, were identified. QYrst.wgp-6BS.1 was within a 3.9-cM region flanked by Xbarc101 and Xbarc136. QYrst.wgp-6BS.2 was mapped in a 17.5-cM region flanked by Xgwm132 and Xgdm113. Both two QTL were physically mapped to the short arm of chromosome 6B, but in different bins. Validation and polymorphism tests of the flanking markers in 43 wheat genotypes indicated that the molecular markers associated with these QTL should be useful in marker-assisted breeding programs to efficiently incorporate HTAP resistance into new wheat cultivars.

Ectopic expression of doublecortin protects adult rat progenitor cells and human glioma cells from severe oxygen and glucose deprivation.

Doublecortin (DCX) is a microtubule-associated protein expressed in migrating neuroblasts. DCX expression is increased in subventricular zone (SVZ) cells migrating to the boundary of an ischemic lesion after induction of middle cerebral artery occlusion (MCAO) in adult rats and mice. We tested the hypothesis that DCX, in addition to being a marker of migrating neuroblasts, serves to protect neuroblasts from conditions of stress, such as oxygen and glucose deprivation (OGD). Using gene transfer technology, we overexpressed DCX in rat SVZ and U-87 human glioma cells. The cells remained viable against severe OGD, up to 32 h exhibiting 1% apoptosis compared with 100% apoptosis in control. In addition, these genetically modified cells upregulated expression of E-, VE- and N-cadherin, molecules that promote endothelial survival signals via the VE-cadherin/vascular endothelial growth factor receptor-2/phosphoinositide 3-kinase (PI3-K)/AKT/beta-catenin pathway and inactivate the proapoptotic factor Bad. DCX overexpression also significantly increased cell migration in SVZ tissue explants and U-87 cells and significantly upregulated microtubule-associated protein-2 (MAP2) and nestin protein levels in SVZ and U-87 cells compared with wild-type control cells. Knocking down DCX expression in DCX overexpressing SVZ and U-87 cells with DCX small interfering RNA (siRNA), confirmed the specificity of DCX on cell survival against OGD, and the DCX induced upregulation of E-, VE- and N-cadherin, MAP2 and nestin. In NIH3T3 cells, DCX overexpression had no effect on cell survival against OGD, and indicating that the protective effects of DCX was restricted to brain cells e.g. SVZ and U-87 cells. Our data suggest a novel and an important role for DCX as a protective agent for migrating neuroblasts and tumor cells.

A composite element binding the vitamin D receptor and the retinoic X receptor alpha mediates the transforming growth factor-beta inhibition of decorin gene expression in articular chondrocytes.

Decorin, a small leucine-rich proteoglycan may play an important role in the attempt of cartilage repair initiated by chondrocytes in early stages of osteoarthritis, through its ability to bind collagen fibrils and growth factors such as transforming growth factor-beta (TGF-beta). We previously demonstrated that TGF-beta decreased decorin mRNA steady state levels in articular chondrocytes (Demoor, M., Rédini, F., Boittin, M., and Pujol, J.-P. (1998) Biochim. Biophys. Acta 1398, 179-191). Here, we investigated the effect of TGF-beta on decorin gene expression in both primary cultures of articular chondrocytes and chondrocytes dedifferentiated by serial passages. Transient transfection of cells with plasmid constructs of the decorin promoter linked to the luciferase reporter gene revealed transcriptional repression by TGF-beta, in fully differentiated as well as dedifferentiated chondrocytes. Experiments with 5'-deleted constructs allowed characterization of a TGF-beta-responsive element in the shortest construct (base pairs (bp) -155/+269). DNase I footprinting analysis delineated a negative TGF-beta-responsive region between -140 and -111 bp in the decorin proximal promoter. Gel retardation assays demonstrated that TGF-beta modulates decorin gene expression through transcription factors, the nature and mode of action of which depend on the differentiation state of the chondrocytes; two DNA-protein complexes were formed in the region -144/-127 bp with nuclear extracts from primary chondrocytes, whereas a higher mobility complex was observed in the -127/-111 bp region for dedifferentiated cells. Antibodies against vitamin D and retinoic acid receptors used in supershift experiments showed that these nuclear receptors are involved in the regulation of decorin gene expression in articular chondrocytes.

An anti-oncogenic role for decorin. Down-regulation of ErbB2 leads to growth suppression and cytodifferentiation of mammary carcinoma cells.

The leucine-rich proteoglycan decorin interacts with the epidermal growth factor receptor and triggers a signaling pathway that leads to growth suppression. We find that decorin causes a functional inactivation of the oncogenic ErbB2 protein in breast carcinoma cells. Upon de novo expression of decorin, the ErbB2 protein is reduced by approximately 40%, whereas its degree of tyrosyl phosphorylation is almost completely abrogated. Both co-culture experiments or experiments with recombinant decorin demonstrate an initial induction of ErbB2 tyrosine kinase, followed by a profound and long-lasting down-regulation of its activity. This leads to growth inhibition and cytodifferentiation of mammary tumor cells and a concurrent suppression of their tumorigenic potential in vivo. These decorin-mediated effects appear to involve the activation of ErbB4, which in turn would block the phosphorylation of heterodimers containing either ErbB2 or ErbB3. These results provide an explanation for the heightened decorin levels around invasive carcinomas and suggest that decorin may function as a natural antagonist of neoplastic cells enriched in ErbB2.

Sustained down-regulation of the epidermal growth factor receptor by decorin. A mechanism for controlling tumor growth in vivo.

The small leucine-rich proteoglycan decorin interacts with the epidermal growth factor receptor (EGFR) and triggers a signaling cascade that leads to elevation of endogenous p21 and growth suppression. We demonstrate that decorin causes a sustained down-regulation of the EGFR. Upon stable expression of decorin, the EGFR number is reduced by approximately 40%, without changes in EGFR expression. However, EGFR phosphorylation is nearly completely abolished. Concurrently, decorin attenuates the EGFR-mediated mobilization of intracellular calcium and blocks the growth of tumor xenografts by down-regulating the EGFR kinase in vivo. Thus, decorin acts as an autocrine and paracrine regulator of tumor growth and could be utilized as an effective anti-cancer agent.

Activation of extracellular signal-regulated protein kinase1,2 results in down-regulation of decorin expression in fibroblasts.

Decorin is a small leucine-rich extracellular matrix proteoglycan, the expression of which is down-regulated in proliferating and malignantly transformed cells. In the present study we show that the expression of decorin in fibroblasts is suppressed by epidermal growth factor (EGF) and PMA, and that the effect of both is potently inhibited by blocking the extracellular signal-regulated protein kinase (ERK)1,2 signalling pathway (Raf/MEK1,2/ERK1,2) with the specific MAPK/ERK kinase (MEK)1,2 inhibitor, PD98059. In addition, specific activation of ERK1,2 by adenovirus-mediated expression of constitutively active MEK1 in dermal fibroblasts results in marked reduction in decorin mRNA abundance and production. Co-transfection of NIH-3T3 fibroblasts with human decorin promoter/chloramphenicol acetyltransferase (CAT) construct (pDEC--879/CAT) in combination with the expression vectors for constitutively active Raf-1 and MEK1 markedly suppressed decorin promoter activity. Co-transfections of human decorin promoter 5'-deletion constructs with constitutively active MEK1 expression vector identified the region -278 to -188 as essential for ERK1,2 mediated down-regulation of decorin promoter activity. These results show that activation of the ERK1,2 signalling pathway by a mitogenic growth factor, a tumour promoter or transformation suppresses decorin gene expression in fibroblasts, which in turn may promote proliferation and migration of normal and malignant cells.

Clastogenic effects of zinc chloride on human peripheral blood leucocytes in vitro.

The clastogenic effects of three different concentrations of zinc chloride on human peripheral blood leucocytes were studied in vitro. The highest concentration (1.5 x 10(-3) M) was lethal after 48 and 72 h of culture and no blast cells were formed. The two lower concentrations (3.0 x 10(-4) M and 3.0 x 10(-5) M) significantly reduced the frequency of cell division, induced chromatid breaks and damaged cells in frequencies significantly higher than in control experiments maintained in sodium chloride and in distilled water.

Comparison of chromosome damage induced by three zinc compounds using human leukocyte culture.

The degree of chromosome damage induced by three compounds of zinc (zinc chloride, zinc sulfate, zinc acetate) was compared in human leucocytes in vitro. Three concentrations of each salt, 3.0 x 10(-5)M, 3.0 x 10(-4)M, and 1.5 x 10(-3)M, were added to leukocyte cultures. The cells were harvested after 48 and 72 h and chromosome spreads were prepared following a colchicine-hypotonic-fixation-air drying-Giemsa staining schedule. The end point screened was chromosome aberrations. All three salts were lethal at the highest concentration. The degree of chromosome damage was directly proportional to the concentrations used for zinc sulfate and zinc acetate but not for zinc chloride.

Decorin activates the epidermal growth factor receptor and elevates cytosolic Ca2+ in A431 carcinoma cells.

Several independent lines of evidence have implicated decorin, a small leucine-rich proteoglycan, in the inhibition of cell proliferation. However, the mechanism by which decorin mediates its effect on cell proliferation is unclear. Here we report, for the first time, decorin-mediated increases in intracellular Ca2+ levels of single A431 cells. The effects of decorin persisted in the absence of extracellular Ca2+ but were blocked by AG1478, an epidermal growth factor (EGF)-specific tyrosine kinase inhibitor, and by down-regulation of the EGF receptor. The effects of decorin were not mimicked by the structurally homologous protein, biglycan. Our results indicate a novel action of decorin on the EGF receptor, which results in mobilization of intracellular Ca2+ providing a possible mechanism by which decorin causes growth suppression.

Decorin suppresses tumor cell growth by activating the epidermal growth factor receptor.

Decorin, a small leucine-rich proteoglycan, is capable of suppressing the growth of various tumor cell lines when expressed ectopically. In this report, we investigated the biochemical mechanism by which decorin inhibits cell cycle progression. In A431 squamous carcinoma cells, decorin proteoglycan or protein core induced a marked growth suppression, when either exogenously added or endogenously produced by a transgene. Decorin caused rapid phosphorylation of the EGF receptor and a concurrent activation of mitogen-activated protein (MAP) kinase signal pathway. This led to a protracted induction of endogenous p21, a potent inhibitor of cyclin-dependent kinases, and ultimate cell cycle arrest. Biglycan, a related proteoglycan, had no effect. Moreover, decorin activated the EGF receptor/MAP kinase/ p21 axis in cell lines of various histogenetic backgrounds. These results provide the first evidence that EGF and decorin converge functionally to regulate the cell cycle through activation of a common pathway which ultimately leads to growth suppression.

Ectopic expression of decorin protein core causes a generalized growth suppression in neoplastic cells of various histogenetic origin and requires endogenous p21, an inhibitor of cyclin-dependent kinases.

Decorin belongs to a family of secreted, small, leucine-rich proteoglycans that affect matrix assembly and cellular growth. Ectopic expression of decorin proteoglycan or protein core as a mutated form lacking any glycosaminoglycan side chains induced growth suppression in neoplastic cells of various histogenetic origins, including tumor cells derived from gastrointestinal, genital, skeletal, cutaneous, or bone marrow tissues. Exogenously added recombinant decorin also suppressed overall growth of the parental cell lines. In all stably-transfected clones, growth retardation was specifically associated with induction of the potent cyclin-dependent kinase inhibitor p21, but not p27, and subsequent translocation of p21 protein into the nuclei of decorin-expressing cells. This led to a greater proportion of the cells arrested in G1 phase of the cell cycle. These changes were independent of functional p53 or retinoblastoma protein. De novo expression of decorin in HCT116 human colon carcinoma cells harboring a disrupted p21 gene failed to induce growth suppression, in contrast to the wild-type cells in which p21 and growth arrest could be induced. These findings indicate that ectopic production of decorin protein core can retard the growth of a variety of tumor cells and that endogenous p21 is a required downstream effector of this biological axis.

Identification of a bimodal regulatory element encompassing a canonical AP-1 binding site in the proximal promoter region of the human decorin gene.

We have previously described a tumor necrosis factor alpha (TNF-alpha) response element, located between residues -188 and -140 of the human decorin promoter, that mediates the inhibitory effect of TNF-alpha on decorin gene expression (Mauviel, A., Santra, M., Chen, Y.-Q., Uitto, J., and Iozzo, R. V. (1995) J. Biol. Chem. 270, 11692-11700). In this report, we demonstrate that interleukin 1 (IL-1), a pleiotropic cytokine that shares a wide variety of biological properties with TNF-alpha, uses the same cis element to up-regulate decorin gene expression. Specifically, IL-1 enhances the expression of the human decorin gene, and this effect is mediated by activation of the corresponding promoter, as shown in transient cell transfection experiments using decorin promoter-chloramphenicol acetyl transferase reporter gene constructs. Additional transfection experiments with various 5'-deletion promoter-chloramphenicol acetyltransferase constructs demonstrate that both the inhibitory effect of TNF-alpha and the stimulatory effect of IL-1 are mediated by a 48-base pair segment of the promoter, between residues -188 and -140. This region, which contains a canonical AP-1 binding site, TGAGTCA, allows an antagonistic effect of these two cytokines on the decorin promoter activity. When cloned upstream of the thymidine kinase promoter, this promoter fragment requires the AP-1 sequence to be responsive to IL-1. Supershift assays with various AP-1 antibodies identified c-Jun, Jun-B, and Fra-1 as components of the complex binding to the decorin promoter. Overexpression of c-jun, an oncogene encoding the c-Jun/AP-1 transcription factor, reduces the basal activity of both decorin and -188/-140 thymidine kinase promoter constructs. In contrast, blockage of c-jun expression with an antisense c-jun construct potentiates the stimulatory effect of IL-1 and reverses the response to TNF-alpha. These data indicate that the region between residues -188 and -140 of the human decorin promoter functions as a bimodal regulatory element and allows transcriptional repression by c-Jun/AP-1 complexes.

Decorin-induced growth suppression is associated with up-regulation of p21, an inhibitor of cyclin-dependent kinases.

The secreted proteoglycan decorin has been implicated in the negative control of cell proliferation primarily by virtue of its ability to block transforming growth factor-beta. Moreover, decorin expression is markedly up-regulated during quiescence but suppressed upon viral transformation, whereas de novo decorin expression in colon carcinoma cells abrogates the malignant phenotype by arresting the cells in the G1 phase of the cell cycle. Here we show that this decorin-induced growth arrest is associated with up-regulation of p21 mRNA and protein in a transforming growth factor-beta- and p53-independent pathway. The augmented p21 protein is present as a multimeric complex with various cyclins and cyclin-dependent kinases in the nuclei of decorin-expressing cells, thereby leading to suppression of cyclin-dependent kinase activity and block of cell division. Through the usage of decorin-specific antisense oligodeoxynucleotide treatment, we demonstrate that the expression of decorin is closely linked to that of p21 and that abrogation of decorin leads to suppression of p21 and restoration of cell division. Collectively, our results provide a plausible mechanism by which decorin may contribute to retard and suppress the growth of tumor cells in vivo.

De novo decorin gene expression suppresses the malignant phenotype in human colon cancer cells.

The rapid progress in the cloning of proteoglycan genes has enabled investigators to examine in depth the functional roles these polyhedric molecules play in the control of cell proliferation. Decorin, a leucine-rich proteoglycan expressed by most connective tissues, is a prototype molecule that regulates cellular growth via two mechanisms: modulation of growth factor activity and matrix assembly. We now provide direct evidence that human colon cancer cells stably transfected with decorin cDNA exhibit a marked suppression of the transformed phenotype: the cells have a reduced growth rate in vitro, form small colonies in soft agar, and do not generate tumors in scid/scid mice. Several independent clones are arrested in the G1 phase of the cell cycle, and their growth suppression can be restored by treatment with decorin antisense oligodeoxynucleotides. These effects are independent of growth factors and are not due to either clonal selection or integration site of the decorin gene. These findings correlate well with the observation that decorin gene expression is markedly up-regulated during quiescence. Decorin thus appears to be one component of a negative loop that controls cell growth.

Transcriptional regulation of decorin gene expression. Induction by quiescence and repression by tumor necrosis factor-alpha.

Decorin, a leucine-rich proteoglycan with ubiquitous tissue distribution, may play essential biological roles during inflammation and cancer growth through its ability to bind extracellular matrix constituents and growth factors. In this study, we demonstrate that decorin gene expression is greatly enhanced after normal diploid fibroblasts reach confluency and cease to proliferate. Elevation of decorin mRNA steady state levels was maintained for up to 16 days postconfluency. In vitro transcription analyses indicated enhanced transcriptional activity in quiescent fibroblasts when compared to cells harvested in their logarithmic phase of growth. This phenotypic trait was reversed by the exogenous addition of tumor necrosis factor-alpha (TNF-alpha). Furthermore, transforming growth factor-beta (TGF-beta) down-regulated decorin gene expression in an additive manner with TNF-alpha. Transient cell transfection assays using plasmid constructs harboring the decorin promoter linked to the chloramphenicol acetyltransferase reporter gene demonstrated a dose-dependent transcriptional repression by TNF-alpha. These findings were further corroborated by in vitro transcription experiments using nuclear extracts from control and TNF-alpha-treated quiescent fibroblasts. In contrast, the decorin promoter constructs failed to respond to TGF-beta, thus suggesting either post-transcriptional regulation by this growth factor or lack of TGF-beta-responsive elements. Further experiments with 5' deletion constructs showed two TNF-alpha response elements, one residing within the 5'-untranslated region (exon Ib), the other one between residues -188 and -140 of the decorin promoter. Collectively, our results indicate that TNF-alpha, through its ability to transcriptionally inhibit decorin gene expression in growth-arrested cells, may be a key modulator of the biological functions of this proteoglycan.

Structural and functional characterization of the human decorin gene promoter. A homopurine-homopyrimidine S1 nuclease-sensitive region is involved in transcriptional control.

Decorin is a leucine-rich, chondroitin/dermatan sulfate proteoglycan which binds collagen and growth factors. We have recently completed the genomic organization of human decorin and discovered two alternatively spliced leader exons, designated exon Ia and Ib, in the 5'-untranslated region. Initial analysis of the sequences upstream to these two exons showed that promoter Ia contained only two GC boxes while promoter Ib contained a CAAT and two TATA boxes in close proximity to the transcription start site. To determine if these 5'-flanking sequences exhibited promoter activity, chimeric chloramphenicol acetyltransferase expression plasmids containing the promoter region of either exon Ia or Ib were transfected into HeLa and MG-63 osteosarcoma cells. The results showed that only the region flanking exon Ib was functional. In vitro transcription assay generated two transcripts of 92 and 82 base pairs (bp) indicating that both TATA boxes could be used. Using stepwise 5' deletion analysis we found that the minimum promoter region at -140 bp from the transcription start site, which contained only the CAAT and the two TATA boxes, exhibited strong promoter activity. When a larger construct containing an additional 800 bp of upstream region was tested, a significant increase in transcriptional activity was observed. Interestingly, this promoter region contained several putative binding sites for ubiquitous factors (AP1, AP5, and NF-kappa B) and for transforming growth factor-beta and a 150-bp homopurine/homopyrimidine element with several mirror repeats. When contained in a supercoiled plasmid, this sequence exhibited sensitivity to endonuclease S1, an enzyme that preferentially digests single-stranded DNA. Precise S1 mapping, obtained by direct sequencing of nine distinct S1-generated clones, revealed that in all cases the borders of the sensitive sequence resided within the pur/pyr segment. We propose that this region of the promoter could adopt an intramolecular hairpin triplex structure in vivo and may play a role in the chromatin organization at the decorin gene locus. In addition, this region was able to up-regulate a minimal heterologous promoter in transient transfection assays. The results show that the structure of the decorin gene promoter is different from that of any other proteoglycan promoter characterized so far and indicate that the pur/pyr segment plays a role in the regulation of gene transcription.

Mode of action of iron on arylsulphatases under acute lead acetate treated conditions in rats.

Arylsulphatases A, B and C were found to be inhibited in liver and kidney tissues under lead acetate-treated conditions (both in vivo and in vitro) in rats. When lead acetate-treated animals (in vivo) were supplemented with ferric ammonium citrate (in vivo), a remarkable recovery was found in the activities of all arylsulphatases A, B and C whereas ferric ammonium citrate itself had no effect on the activities of arylsulphatases. When both the in vivo and in vitro lead acetate-treated arylsulphatases were supplemented with the purified ferritins (in vitro) it was observed that lead-induced inhibition of the activities of arylsulphatases was successfully reversed. It was also found that ferritins were able to bind a large quantity of lead. These results indicated that ferritins were directly involved for reactivation of arylsulphatases which were inhibited by lead. It was well established that a response to iron administration in rats was an immediate de novo stimulation of ferritin biosynthesis. Iron might therefore protect the enzymatic activities of arylsulphatases by enhancing the level of ferritin in liver and kidney tissues which is known to bind a large quantity of lead thereby ameliorating their toxic effects in the living system.