Trabecular bone quality is lower in adults with type 1 diabetes and is negatively associated with insulin resistance.

We evaluated trabecular bone score (TBS) and factors affecting TBS in adults with type 1 diabetes (T1D) compared to age-, sex-, and body mass index (BMI)-matched adults without diabetes. Adults with T1D had lower TBS compared to controls. Abdominal obesity and insulin resistance are associated with lower TBS. INTRODUCTION: We evaluated TBS, a non-invasive method to evaluate trabecular bone quality at the lumbar spine, in adults with T1D compared to age-, sex-, and BMI-matched adults without diabetes. METHODS: We calculated TBS from adults with T1D (n = 47) and controls (n = 47) who had a lumbar spine dual x-ray absorptiometry (DXA) at their third visit (2006-2009) of the ongoing "Coronary Artery Calcification in Type 1 Diabetes (CACTI) Study." The linear relationships of TBS and bone mineral density (BMD) with hemoglobin A1c, blood pressure, lipids, and insulin resistance were evaluated using Pearson's correlation coefficient. Multiple linear regression was used to test the association of TBS with sex and diabetes while adjusting for other potential confounders. RESULTS: TBS was significantly lower in adults with T1D compared to controls (1.42 ± 0.12 vs 1.44 ± 0.08, p = 0.02) after adjusting for age, sex, current smoking status, and lumbar spine BMD, despite no difference in lumbar spine BMD between the groups. Components of the metabolic syndrome, including diastolic blood pressure, BMI, triglycerides, and insulin resistance were negatively correlated with TBS among patients with T1D. CONCLUSION: Trabecular bone score, an indirect measurement of trabecular bone quality, was lower in adults with T1D compared to controls. Components of metabolic syndrome and insulin resistance were associated with lower TBS in adults with T1D.

Glycaemic variability is associated with coronary artery calcium in men with Type 1 diabetes: the Coronary Artery Calcification in Type 1 Diabetes study.

AIMS: We investigated coronary artery calcium in association with glucose levels and variability measured using continuous glucose monitoring in adults with Type 1 diabetes in the Coronary Artery Calcification in Type 1 Diabetes study. METHODS: Coronary artery calcium was measured by electron beam tomography. The presence of any coronary artery calcium was analysed with respect to glucose levels [mean(T) (mean glucose), % of values < 3.9 mmol/l, > 10 mmol/l and either < 3.9 or > 10 mmol/l] and glycaemic variability [sd(T) (sd of all glucose values); sd(dm) (sd of the daily mean glucose levels) and sd(hh:mm) (glucose sd for a specified time of day, over all days)] using 3-5 days of continuous glucose monitoring from 75 subjects (45 women, 30 men), age 42 ± 9 years (mean ± sd) and diabetes duration of 29 ± 8 years using logistic regression. RESULTS: We observed significant associations between coronary artery calcium and mean(T) (OR = 4.4, 95% CI 1.1-18.6), % of values > 10 mmol/l (OR = 5.5, 95% CI 1.3-22.6), % of measures < 3.9 or > 10 mmol/l (OR = 5.7, 95% CI 1.3-24.9), sd(T) (OR = 4.7, 95% CI 1.1-19.7), sd(dm) (OR = 6.0, 95% CI 1.2-30.4) and sd(hh:mm) (OR = 4.0, 95% CI 1.1-15.4), among men, but none of these variables were associated with the presence of coronary artery calcium in women. CONCLUSIONS: We report the novel finding that subclinical atherosclerosis is associated with glucose levels and variability in men with Type 1 diabetes. The relationship of coronary artery calcium and glucose variability in Type 1 diabetes, and potential gender differences in this association, deserve further study.

Cyclin D1 as a proliferative marker regulating retinoblastoma phosphorylation in mouse lung epithelial cells.

Elevations in cyclin D1 content increase the phosphorylation status of retinoblastoma (Rb) protein to encourage cell cycle transit. We sought to determine if cyclin D1 content could be used as an index of cell proliferation in mouse lung epithelia following growth manipulations in vitro and in vivo. Rb protein concentration was high in 82-132 and LM2, two fast-growing neoplastic mouse lung epithelial cell lines. The hyperphosphorylated form of Rb predominated in these two cell lines, while Rb in slower-growing cell lines was predominantly hypophosphorylated. Consistent with this, more cyclin D1 protein was expressed in the fast-growing cell lines than in slower-growing cells. We therefore tested whether cyclin D1 content varied with growth status. The amount of cyclin D1 decreased upon serum removal coincident with growth inhibition and then increased upon serum re-addition which stimulated resumption of proliferation. This correlation between cyclin D1 content and growth status also occurred in vivo. Cyclin D1 content increased when lungs underwent compensatory hyperplasia following damage caused by butylated hydroxytoluene administration to mice and in lung tumor extracts as compared with extracts prepared from uninvolved tissue or control lungs. We conclude that elevated cyclin D1 levels account, at least in part, for the hyperphosphorylation of Rb in neoplastic lung cells, and are associated with enhanced lung growth in vitro and in vivo.

Oligomers of the Cdc7/Dbf4 protein kinase exist in the yeast cell.

Cdc7/Dbf4 protein kinase is required for the initiation of DNA replication in Saccharomyces cerevisiae. Cdc7/Dbf4 protein kinase is not a cyclin-dependent kinase (CDK), but is regulated in a similar fashion in that the Cdc7 kinase subunit is inactive in the absence of the regulatory subunit Dbf4. In contrast to what is known about CDKs, Cdc7/Dbf4 protein kinase is shown to be an oligomer in the cell in this report. Genetic data that support this claim include interallelic complementation between several cdc7ts alleles and the cdc7T281A allele and also the results of experiments using the two-hybrid system with Cdc7 in both DNA-binding and transactivation domain plasmids. A molecular interaction between two different Cdc7 molecules was shown by using a HA-tagged Cdc7 protein that differs in size from the wild-type Cdc7 protein: an anti-HA antibody immunoprecipitates both proteins in approximately equal stoichiometry. Analysis of the native molecular weight of Cdc7/Dbf4 protein kinase is consistent with oligomerization of the Cdc7 protein in that complexes of about 180 and 300 kDa were found. Oligomers of Cdc7 protein may exist for the purpose of allosteric regulation or to allow phosphorylation of multiple substrate protein molecules.

Biphasic regulation of breast cancer cell growth by progesterone: role of the cyclin-dependent kinase inhibitors, p21 and p27(Kip1).

Depending on the tissue, progesterone is classified as a proliferative or a differentiative hormone. To explain this paradox, and to simplify analysis of its effects, we used a breast cancer cell line (T47D-YB) that constitutively expresses the B isoform of progesterone receptors. These cells are resistant to the proliferative effects of epidermal growth factor (EGF). Progesterone treatment accelerates T47D-YB cells through the first mitotic cell cycle, but arrests them in late G1 of the second cycle. This arrest is accompanied by decreased levels of cyclins D1, D3, and E, disappearance of cyclins A and B, and sequential induction of the cyclin-dependent kinase (cdk) inhibitors p21 and p27(Kip1). The retinoblastoma protein is hypophosphorylated and extensively down-regulated. The activity of the cell cycle-dependent protein kinase, cdk2, is regulated biphasically by progesterone: it increases initially, then decreases. This is consistent with the biphasic proliferative increase followed by arrest produced by one pulse of progesterone. A second treatment with progesterone cannot restart proliferation despite adequate levels of transcriptionally competent PR. Instead, a second progesterone dose delays the fall of p21 and enhances the rise of p27(Kip1), thereby intensifying the progesterone resistance in an autoinhibitory loop. However, during the progesterone-induced arrest, the cell cycling machinery is poised to restart. The first dose of progesterone increases the levels of EGF receptors and transiently sensitizes the cells to the proliferative effects of EGF. We conclude that progesterone is neither inherently proliferative nor antiproliferative, but that it is capable of stimulating or inhibiting cell growth depending on whether treatment is transient or continuous. We also suggest that the G1 arrest after progesterone treatment is accompanied by cellular changes that permit other, possibly tissue-specific, factors to influence the final proliferative or differentiative state.

Cell cycle control and cancer: lessons from lung cancer.

Recent developments in the study of the cell cycle have shed much light on the origins of human cancer. We summarize these developments with an emphasis on the molecular characterization and the functional role of the cyclin-dependent kinase family of protein kinases (CDK) and their associated regulatory subunits. The Rb tumor suppressor in the progression from the G1 to S phase of the cell cycle and in tumor development is used as a paradigm for illustrating the importance of understanding the molecular regulatory events in the etiology of cancer. Recent developments with cyclin-dependent kinase inhibitors, most notably, p16 (CDKN2), indicate that these molecules represent new tumor suppressors in both skin and lung cancers. Insights from these cell cycle studies can provide avenues for the diagnosis, prognosis, and potential gene and chemotherapies for many cancers, including non-small cell lung cancer.

Cyclin D1 overexpression vs. retinoblastoma inactivation: implications for growth control evasion in non-small cell and small cell lung cancer.

The cyclin-dependent kinases and their associated regulatory cyclins control cell cycle progression and cell growth. Antibodies against these proteins were used to determine their levels in several lung tumor-derived cell lines and a "normal" immortalized bronchoepithelial cell line in order to investigate their potential roles in the etiology of lung cancer. All the cell lines expressed roughly equal levels of cdk-1; cdk-2; PSTAIRE-sequence containing kinases; proliferating cell nuclear antigen; and cyclins A, B1, and E. Cyclin D1, however, was present at 4- to 100-fold higher levels in 11 of 12 non-small cell lung cancer cell lines than in the bronchoepithelial line and all but one of the small cell lung cancer lines. Furthermore, immunoblots of the retinoblastoma gene product, pRB, revealed a perfect correlation between pRB levels and tumor type with normal levels of phosphorylation-competent pRB in all of the non-small cell lung cancer lines and undetectable levels of pRB in all of the small cell lung cancer lines. These data suggest the possibility that small cell and non-small cell lung cancer may evade normal growth controls by different mechanisms: loss of the proliferation inhibitor pRB in small cell lung cancer and overexpression of the growth promoting cyclin D1 in non-small cell lung cancer.

Early C. elegans embryos are transcriptionally active.

We have developed a nucleotide incorporation assay for run-on transcription in C. elegans embryonic extracts as an approach to characterizing early transcription. The incorporation is primarily polymerase II-catalyzed RNA synthesis, producing transcripts of the expected size range for mRNAs. Incorporation is insensitive to inhibitors of reinitiation, indicating that the activity represents primarily elongation of nascent chains initiated prior to extract preparation. The transcripts produced appear to be unprocessed pre-mRNAs. Hybridization of labeled transcripts from extracts of staged embryos to a set of cloned genes suggests that the specificity of the in vitro reaction accurately reflects developmentally regulated in vivo transcription. Comparative analyses of transcription in extracts from various stages indicate that pregastrulation embryos are active transcriptionally and that the level of transcription per nucleus is approximately constant throughout embryogenesis. Furthermore, most embryonically expressed genes are already being transcribed in pregastrulation embryos. We also demonstrate that the labeled embryonic run-on transcripts can be used as probes to screen for sequences transcribed preferentially in pregastrulation embryos. There appears to be only a small set of such sequences, which could represent a previously unsuspected class of embryonically transcribed genes important for early embryogenesis.

Structure, assembly, and secretion of octameric invertase.

Yeast invertase forms a homo-octamer of core glycosylated subunits during assembly in the lumen of the endoplasmic reticulum. This form has been purified from mutant cells (sec18) in which transport of secreted proteins from the endoplasmic reticulum is blocked. No heterologous protein subunits are found in the purified material. Analysis of invertase derived from wild type cells or from mutant cells blocked at subsequent stages in secretion demonstrates that invertase remains a homo-octamer throughout the pathway even though the extent of subunit glycosylation increases. Purified octameric invertase is dissociated into dimer units that reassociate in the presence of polyethylene glycol. Negatively stained preparations show the dissociated enzyme as individual spheres, whereas octameric invertase appears as four associated spheres. Assembly of the octamer in vitro and in vivo is facilitated by the presence of N-linked carbohydrate. Selective release of dimeric glycosylated invertase from intact yeast cells suggests that oligomerization helps retain the enzyme in the periplasmic space.

Isolation of hypoxanthine phosphoribosyltransferase-defective mutants in Chinese hamster V79 cells by tritium suicide.

Tritium suicide was shown to be a highly efficient method for isolating mutants defective in hypoxanthine incorporation in the Chinese hamster lung cell line V79. The tritium suicide procedure consisted of 3 kill cycles. Survivors of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of [3H]hypoxanthine for 5 or 10 min, followed by storage of 3H-labelled cells at -70 degrees C for 4-10 days. 12 clones that survived the 3rd kill cycle were tested for incorporation of [3H]hypoxanthine and all were found to be defective. At lest 6 of the clones have defective hypoxanthine phosphoribosyltransferase (HPRT) activity. One mutant, H19, chosen for further characterization, had HPRT with a 13-fold elevation in apparent Km for phosphoribosylpyrophosphate (PRPP). Thin-layer chromatography of cell extracts showed that this mutant was incapable of converting intracellular hypoxanthine to IMP or to other purine metabolites. In addition, H19 as resistant to 6-thioguanine.