Kumar, Rajiv

Abstract When cells encounter substantial DNA damage, critical cell cycle events are halted while DNA repair mechanisms are activated to restore genome integrity. Genomic integrity also depends on proper assembly and function of the bipolar mitotic spindle, which is required for equal chromosome segregation. Failure to execute either of these processes leads to genomic instability, aging, and cancer. Here, we show that following DNA damage in the breast cancer cell line MCF-7, the centrosome protein centrin2 moves from the cytoplasm and accumulates in the nucleus in a xeroderma pigmentosum complementation group C protein (XPC)–dependent manner, reducing the available cytoplasmic pool of this key centriole protein and preventing centrosome amplification. MDA-MB 231 cells do not express XPC and fail to move centrin into the nucleus following DNA damage. Reintroduction of XPC expression in MDA-MB 231 cells rescues nuclear centrin2 sequestration and reestablishes control against centrosome amplification, regardless of mutant p53 status. Importantly, the capacity to repair DNA damage was also dependent on the availability of centrin2 in the nucleus. These observations show that centrin and XPC cooperate in a reciprocal mechanism to coordinate centrosome homeostasis and DNA repair and suggest that this process may provide a tractable target to develop treatments to slow progression of cancer and aging. Cancer Res; 70(8); 3320–8. ©2010 AACR.

Abstract Mammary glands are target tissues for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We have examined a mouse mammary tumor cell line (GR) for receptors of 1,25(OH)2D3 and have examined alterations in the growth and morphology of these cells in response to 1,25(OH)2D3. GR cells contain a high affinity (Kd ∼ 10−11), lowcapacity receptor with a high specificity for 1,25(OH)2D3. The 1,25(OH)2D3 receptor in GR cells has a sedimentation coefficient of 3.5 and elutes from DEAE cellulose columns with ∼ 0.15 M KCI. These properties of the receptor are similar to those reported for other 1,25(OH)2D3 receptors. 1,25(OH)2D3 is internalized by GR cells in situ and specifically bound 1,25(OH)2D3 is found predominantly, if not entirely, in the nucleus as determined by cell fractionation and autoradiographic techniques. The incubation of GR cells in culture for 7 days with 1,25(OH)2D, markedly alters cell growth. Cell growth is retarded in a dose-dependent manner; physiologic concentrations (10−10M) of l,25(OH)2D3 retard cell growth by approximately 50%. In addition, GR cells incubated with 10−9 to 10−8M 1,25(OH)2D3 undergo marked morphological changes. The incubation of GR cells with other vitamin D metabolites such as 25-hydroxyvitamin D3 (25(OH)D3) at a concentration of 10−9M does not significantly alter cell growth or morphology. The presence of high affinity receptors for 1,25(OH)2D3, the specific internalization of 1,25(OH)2D3 predominantly into the nuclei, and the significant effects of physiological concentrations of 1,25(OH)2D3 on cell growth suggest a direct, specific, nuclear effect of 1,25(OH)2D3 on GR cells. The mouse mammary tumor model might be useful in examining the effect of 1,25(OH)2D3 on tumor formation.

Abstract We studied the mechanism of impaired calcium absorption with aging in 51 healthy women whose ages ranged from 26 to 88 years. Serum concentrations of 1,25-dihydroxyvitamin D [1,25-(OH)2D, mean of four measurements per subject] increased with age by 22% (P < 0.05) but, by split-point analysis, plateaued or decreased slightly after age 65. In a subset of 20 subjects, [3H]1,25-(OH)2D3 kinetic analysis showed that this increase with age resulted from both increased production and decreased metabolic clearance of 1,25-(OH)2D. Despite the increase in serum 1,25-(OH)2D concentration, true calcium absorption did not change with age. The expected inverse correlation between true fractional calcium absorption and dietary calcium intake, however, was easily demonstrated (r = 0.66, P < 0.001). Serum intact parathyroid hormone (PTH) increased with age by 35% (P < 0.02) and serum bone gla protein (BGP, osteocalcin) increased by 47% (P < 0.001); the increases in serum PTH and serum BGP were directly correlated (r = 0.32, P < 0.05). The data are consistent with the following hypothetical model: (1) intestinal resistance to 1,25-(OH)2D action accounts for the increase in serum 1,25-(OH)2D concentrations with aging with no change in true calcium absorption; (2) this results in a compensatory increase in PTH secretion and in 1,25-(OH)2D production that prevents true calcium absorption from decreasing; (3) the previously described defect in 25-hydroxyvitamin D (25-OHD) 1α-hydroxylase activity in aging animals and humans acccounts for the leveling off in serum 1,25-(OH)2D concentration after age 65 years; and (4) the secondary hyperparathyroidism leads to increased bone turnover and thus contributes to age-related bone loss.

Abstract The osteoblast plays a critical role in bone formation, bone remodeling, bone matrix formation, and matrix calcification. To better understand the process of osteoblast-controlled bone formation, we determined the structure and isoform types of the plasma membrane calcium pump from normal human osteoblasts. A complementary DNA library from normal human osteoblasts was screened for plasma membrane calcium pump clones. Sequencing and analysis of cDNA clones revealed the presence of a 3986 base pair cDNA that encoded a 1220 amino acid protein that was similar to the human plasma membrane calcium pump isoform 1. Polyadenylated RNA from human osteoblast cells contains bands of RNA approximately 5050 and 6750 bases long. Reverse transcription of polyadenylated RNA from human osteoblasts followed by amplification of the RNA-DNA duplex with calcium pump isoform-specific primers revealed the presence of isoforms 1 and 2 of the calcium pump. Isoform 4 was not detected. We conclude that normal adult human osteoblasts contain a plasma membrane calcium pump that is similar to the human plasma membrane calcium pump isoform 1. It is likely that this pump plays an important role in the cell biology of the human osteoblast.

Abstract To gain insights into 1,25-dihydroxyvitamin D3 receptor (VDR) function during fetal bone development, we examined fetal rat tissues from gestational days 13–21 for the presence and distribution of VDR using immuno-histochemistry. Prior to ossification, VDR epitopes were observed in the mesenchyme condensing to form skeletal tissues, on day 13 in the developing vertebral column and limbs, and on day 17 of gestation in developing calvaria. Immunostaining for VDR was seen in proliferating and hypertrophic chondrocytes and in osteoblasts of limb buds and the vertebral column by day 17 of gestation. In calvaria, VDR epitopes were observed in osteoblasts by gestational day 19. VDR immunostaining was also evident in the skin of fetal limbs at all gestational ages examined. We show for the first time that the VDR appears very early in the developing fetal rat skeleton, suggesting that the VDR, in concert with its ligand, 1,25-dihydroxyvitamin D3, may play a role in the differentiation of mesenchymal precursors into bone tissue.

We developed and characterized monoclonal antibodies directed against the amino-terminal and carboxy-terminal regions of human and mouse sclerostin (scl). Amino-terminal and carboxy-terminal scl peptides with limited homology to scl domain-containing protein-1 were synthesized using f-moc chemistry. The peptides were conjugated to keyhole limpet hemocyanin and the conjugates were used for immunization of mice. Monoclonal antibodies were obtained and characterized using bacterially expressed and insect cell–expressed recombinant scl. The amino-terminal (IgG 2aK) and carboxy-terminal (IgG 2bK) antibodies bound bioactive sclerostin that was expressed in an insect-cell expression system with dissociation constants in the nanomolar range. The antibodies are potentially useful agents that can be used for modulating sclerostin bioactivity.

Abstract IER3 (formerly IEX-1) encodes a 27-kDa glycoprotein that regulates death receptor–induced apoptosis, interacts with NF-κB pathways, and increases expression rapidly in response to cellular stresses such as irradiation. Animal models, gene expression microarray experiments, and functional studies in cell lines have suggested a potential role for IER3 in oncogenesis, but, to date, no abnormalities of IER3 at the DNA level have been reported in patients with neoplasia. Here, we describe breakpoint cloning of a t(6;9)(p21;q34) translocation from a patient with a myelodysplastic syndrome (MDS), facilitated by conversion technology and array-based comparative genomic hybridization, which revealed a rearrangement translocating the IER3 coding region away from critical flanking/regulatory elements and to a transcript-poor chromosomal region, markedly decreasing expression. Using split-signal and locus-specific fluorescence in situ hybridization (FISH) probes, we analyzed 204 patients with diverse hematological malignancies accompanied by clonal chromosome 6p21 abnormalities, and found 8 additional patients with MDS with IER3 rearrangements (translocations or amplification). Although FISH studies on 157 additional samples from patients with MDS and a normal-karyotype were unrevealing, and sequencing the IER3 coding and proximal promoter regions of 74 MDS patients disclosed no point mutations, reverse transcription-PCR results suggested that dysregulated expression of IER3 is common in MDS (61% >4-fold increase or decrease in expression with decreased expression primarily in early MDS and increased expression primarily in later MDS progressing toward leukemia), consistent with findings in previous microarray experiments. These data support involvement of IER3 in the pathobiology of MDS. [Cancer Res 2009;69(19):7518–23]

Abstract Background: Urinary albumin excretion is a sensitive diagnostic and prognostic marker for renal disease. Therefore, measurement of urinary albumin must be accurate and precise. We have developed a method to quantify intact urinary albumin with a low limit of quantification (LOQ). Methods: We constructed an external calibration curve using purified human serum albumin (HSA) added to a charcoal-stripped urine matrix. We then added an internal standard, 15N-labeled recombinant HSA (15NrHSA), to the calibrators, controls, and patient urine samples. The samples were reduced, alkylated, and digested with trypsin. The concentration of albumin in each sample was determined by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and linear regression analysis, in which the relative abundance area ratio of the tryptic peptides 42LVNEVTEFAK51 and 526QTALVELVK534 from albumin and 15NrHSA were referenced to the calibration curve. Results: The lower limit of quantification was 3.13 mg/L, and the linear dynamic range was 3.13–200 mg/L. Replicate digests from low, medium, and high controls (n = 5) gave intraassay imprecision CVs of 2.8%–11.0% for the peptide 42LVNEVTEFAK51, and 1.9%–12.3% for the 526QTALVELVK534 peptide. Interassay imprecision of the controls for a period of 10 consecutive days (n = 10) yielded CVs of 1.5%–14.8% for the 42LVNEVTEFAK51 peptide, and 6.4%–14.1% for the 526QTALVELVK534 peptide. For the 42LVNEVTEFAK51 peptide, a method comparison between LC-MS/MS and an immunoturbidometric method for 138 patient samples gave an R2 value of 0.97 and a regression line of y = 0.99x + 23.16. Conclusions: Urinary albumin can be quantified by a protein cleavage LC-MS/MS method using a 15NrHSA internal standard. This method provides improved analytical performance in the clinically relevant range compared to a commercially available immunoturbidometric assay.

Phosphorus plays a critical role in diverse biological processes, and, therefore, the regulation of phosphorus balance and homeostasis are critical to the well being of the organism. Changes in environmental, dietary, and serum concentrations of inorganic phosphorus are detected by sensors that elicit changes in cellular function and alter the efficiency by which phosphorus is conserved. Short-term, post-cibal responses that occur independently of hormones previously thought to be important in phosphorus homeostasis may play a larger role than previously appreciated in the regulation of phosphorus homeostasis. Several hormones and regulatory factors such as the vitamin D endocrine system, parathyroid hormone, and the phosphatonins (FGF-23, sFRP-4, MEPE) among others, may play a role only in the long-term regulation of phosphorus homeostasis. In this review, we discuss how organisms sense changes in phosphate concentrations and how changes in hormonal factors result in the conservation or excretion of phosphorus.

Abstract Background: The accurate and precise measurement of urinary albumin is critical, since even minor increases are diagnostically sensitive indicators of renal disease, cardiovascular events, and risk for death. To gain insights into potential measurement biases, we systematically compared urine albumin measurements performed by LC-MS, a clinically available immunoturbidimetric assay, and size-exclusion HPLC. Methods: We obtained unused clinical urine samples from 150 patients who were stratified by degrees of albuminuria (<20 mg/L, 20–250 mg/L, >250 mg/L) as determined by the immunoturbidimetric assay used in our clinical laboratory (Roche Hitachi 912). Urine albumin was then remeasured via LC-MS and HPLC (Accumin™) assays. Results: The immunoturbidimetric assay, calibrated using manufacturer-supplied serum-derived calibrators (Diasorin), underestimated albumin compared with LC-MS. After calibration with purified HSA, this immunoturbidimetric assay correlated well with LC-MS. HPLC overestimated albumin compared with both LC-MS and immunoturbidimetry. The current LC-MS and HPLC assays both performed poorly at concentrations <20 mg/L. Conclusions: Efforts are needed to establish gold-standard traceable calibrators for clinical assays. LC-MS is a specific method to quantify albumin in native urine when concentrations exceed 20 mg/L, and therefore could be employed for standardization among assays.