Simulated effects of early menopausal bone mineral density preservation on long-term fracture risk: a feasibility study.

Prevention of early menopausal bone loss may reduce the future burden of osteoporosis. In this modelling exercise, an osteoporosis prevention strategy involving 5-year infusions of zoledronic acid, beginning early in menopause, reduced long-term fracture risk and the proportion of aging women with femoral neck densitometric osteoporosis. This strategy warrants further evaluation. INTRODUCTION: Preventing early menopausal bone loss may substantially reduce the future burden of osteoporosis. We modelled the effects of infrequent zoledronic acid infusions on long-term fracture risk. METHODS: Data from the Canadian Multicentre Osteoporosis Study (CaMos) were used to determine the expected natural history of femoral neck areal bone mineral density (BMD) and fracture risk (using FRAX®) from ages 50-80 for women with no antiresorptive drug exposures. We modelled the effects of three infusions of zoledronic acid (at ages 50, 55, 60) on long-term fracture risk, assuming this intervention would preserve BMD until age 65 years, followed by losses mirroring early menopausal BMD loss. RESULTS: At age 65, untreated women and zoledronic acid recipients had expected mean (SD) femoral neck T-scores of - 1.5(1.0) and - 0.8(1.0), 10-year major osteoporotic fracture (MOF) risks of 9.8%(5.0) and 8.0%(3.7) and hip fracture risks of 1.7%(2.4) and 0.8%(1.2), respectively. At age 80, untreated women and zoledronic acid recipients had expected femoral neck T-scores of - 1.9(0.9) and - 1.4(0.9), MOF risks of 17.9%(8.2) and 14.9%(6.4) and hip fracture risks of 6.3%(6.2) and 4.4%(4.5), respectively. The expected proportion of women with femoral neck T-score ≤ - 2.5 was 14.9% for untreated women and 3.8% for zoledronic acid recipients at age 65, increasing to 28.1% and 12.0%, respectively, at age 80. Numbers-needed-to-treat to prevent one case of densitometric osteoporosis were 9 at age 65 and 5 at age 80. CONCLUSION: Infrequent infusions of zoledronic acid, initiated early in menopause, are expected to reduce long-term fracture risk and result in a substantial reduction in the proportion of women with densitometric osteoporosis after age 65.

Testing a theoretical model of imminent fracture risk in elderly women: an observational cohort analysis of the Canadian Multicentre Osteoporosis Study.

We examined the underlying relationship between fracture risk factors and their imminent risk. Results suggested that having past year fracture, worse past year general health, worse past year physical functioning, and lower past year BMD T-score directly predicted higher imminent fracture risk. Past year falls indirectly predicted imminent risk through physical functioning and general health. INTRODUCTION: This study aimed to examine direct and indirect effects of several factors on imminent (1 year) fracture risk. METHODS: Data from women age 65 and older from population-based Canadian Multicentre Osteoporosis Study were used. Predictors were identified from study years 5 and 10, and imminent fracture data (1-year fracture) came from years 6 and 11 (year 5 predicts year 6, year 10 predicts year 11). A structural equation model (SEM) was used to test the theoretical construct. General health and physical functioning were measured as latent variables using items from the 36-Item Short Form Health Survey (SF-36) and bone mineral density (BMD) T-score was a latent variable based on observed site-specific BMD data (spine L1-L4, femoral neck, total hip). Observed variables were fractures and falls. Model fit was evaluated using root mean square error of approximation (RMSEA), Tucker Lewis index (TLI), and comparative fit index (CFI). RESULTS: The analysis included 3298 women. Model fit tests showed that the SEM fit the data well; χ2(172) = 1122.10 < .001, RMSEA = .03, TLI = .99, CFI = .99. Results suggested that having past year fracture, worse past year general health, worse past year physical functioning, and lower past year BMD T-score directly predicted higher risk of fracture in the subsequent year (p < .001). Past year falls had a statistically significant but indirect effect on imminent fracture risk through physical functioning and general health (p < .001). CONCLUSIONS: We found several direct and indirect pathways that predicted imminent fracture risk in elderly women. Future studies should extend this work by developing risk scoring methods and defining imminent risk thresholds.

The association between sclerostin and incident type 2 diabetes risk: a cohort study.

OBJECTIVE: To determine whether sclerostin is associated with fasting glucose, insulin levels, insulin resistance or increased risk of incident type 2 diabetes. BACKGROUND: Type 2 diabetic patients have a higher risk of fractures. Recent studies suggest sclerostin, a regulator of osteoblast activity, is associated with diabetes. MATERIALS AND METHODS: Sclerostin levels were obtained from 1778 individuals with no history of type 2 diabetes participating in the population-based Canadian Multicentre Osteoporosis Study (CaMos) cohort. Participants were followed until diagnosis of type 2 diabetes, death or end of the study period (31 December 2013). The relationship of sclerostin with fasting glucose, insulin levels and homoeostatic model assessment-insulin resistance (HOMA-IR) was studied in linear regression models. Cox proportional hazards models were used to determine the association of sclerostin levels and the risk of incident type 2 diabetes during a mean 7·5 years of follow-up. RESULTS: Fasting glucose, fasting insulin levels and HOMA-IR were weakly correlated with sclerostin levels (Spearman's correlation coefficient: 0·11, P < 0·05; -0·09, P < 0·05; and -0·07, P = 0·02, respectively). Multiple linear regression analyses confirmed a significant association between sclerostin and fasting insulin and HOMA-IR but no significant association with fasting glucose levels. Sclerostin levels were not found to be significantly associated with the risk of incident type 2 diabetes (HR: 1·30; 95% CI: 0·37-4·57). CONCLUSIONS: We observed an association between sclerostin levels with fasting insulin levels and HOMA-IR, but there was no clear association with type 2 diabetes risk. Further studies are needed to understand the role of sclerostin in type 2 diabetes.

Associations of Protein Intake and Protein Source with Bone Mineral Density and Fracture Risk: A Population-Based Cohort Study.

UNLABELLED: High dietary protein has been hypothesized to cause lower bone mineral density (BMD) and greater fracture risk. Previous results are conflicting and few studies have assessed potential differences related to differing protein sources. OBJECTIVE: To determine associations between total protein intake, and protein intake by source (dairy, non-dairy animal, plant) with BMD, BMD change, and incident osteoporotic fracture. DESIGN/SETTING: Prospective cohort study (Canadian Multicentre Osteoporosis Study). Participants/Measures: Protein intake was assessed as percent of total energy intake (TEI) at Year 2 (1997-99) using a food frequency questionnaire (N=6510). Participants were contacted annually to ascertain incident fracture. Total hip and lumbar spine BMD was measured at baseline and Year 5. Analyses were stratified by group (men 25-49 y, men 50+ y, premenopausal women 25-49 y, and postmenopausal women 50+ y) and adjusted for major confounders. Fracture analyses were limited to those 50+ y. RESULTS: Intakes of dairy protein (with adjustment for BMI) were positively associated with total hip BMD among men and women aged 50+ y, and in men aged 25-49. Among adults aged 50+ y, those with protein intakes of <12% TEI (women) and <11% TEI (men) had increased fracture risk compared to those with intakes of 15% TEI. Fracture risk did not significantly change as intake increased above 15% TEI, and was not significantly associated with protein source. CONCLUSIONS: In contrast to hypothesized risk of high protein, we found that for adults 50+ y, low protein intake (below 15% TEI) may lead to increased fracture risk. Source of protein was a determinant of BMD, but not fracture risk.

Immunohistochemical localisation and effect of matrix metalloproteinases and their inhibitors on canine spontaneous periodontitis.

Periodontitis is commonly observed in dogs. In human medicine, it is well documented that matrix metalloproteinases (MMPs) are involved in the destruction of the periodontium. Therefore, the aim of this prospective study was to investigate the impact of MMPs and their inhibitors, the TIMPs (tissue inhibitors of metalloproteinases), on canine periodontitis. The oral cavities of 57 dogs were examined clinically and radiologically. Gingival biopsies were obtained from the examined dogs and histologically analysed via haematoxylin and eosin stained sections. Immunohistological detection of MMP-2, MMP-3, MMP-8 and MMP-9 as well as TIMP-1 and TIMP-2 was performed by the avidin-biotin peroxidase complex technique. All sections were evaluated by light microscopy. Statistically significant positive correlations were detected between the histologically verified degree of inflammation and the expression of MMP-2, MMP-3, MMP-8 and MMP-9 as well as between changes in collagen fibre content and the occurrence of MMP-2, MMP-8 and MMP-9. Concerning TIMP-1 and TIMP-2, non-significant, generally negative correlations were observed. In summary, in canine periodontitis, an increased expression of the above mentioned MMPs and a tendentially decreased expression of TIMPs are present. In conclusion, in canine periodontitis, a MMP-TIMP imbalance is suggestive of contributing to the destruction of the periodontium.

Characteristics of hyperparathyroid states in the Canadian multicentre osteoporosis study (CaMos) and relationship to skeletal markers.

CONTEXT: PTH is an essential regulator of mineral metabolism; PTH hypersecretion may result in hyperparathyroidism including normocalcaemic, primary and secondary hyperparathyroidism. OBJECTIVE: To examine the characteristics of participants with hyperparathyroid states and the relationship to bone mineral density (BMD). DESIGN AND PARTICIPANTS: A cross-sectional study of 1872 community-dwelling men and women aged 35+ years (mostly Caucasian) with available serum PTH from Year 10 Canadian Multicentre Osteoporosis Study follow-up (2005-07). PTH was determined using a second-generation chemiluminescence immunoassay. OUTCOME MEASURES: L1-L4, femoral neck and total hip BMD. RESULTS: We established a PTH reference range (2·7-10·2 pmol/l) based on healthy participants (i.e. normal serum calcium, serum 25-hydroxyvitamin D, kidney function and body mass index, who were nonusers of antiresorptives, glucocorticoids and diuretics and not diagnosed with diabetes or thyroid disease). Participants with PTH levels in the upper reference range (5·6-10·2 pmol/l), representing a prevalence of 10·7%, had lower femoral neck and total hip BMD, by 0·030 g/cm(2) [95% confidence interval: 0·009; 0·051] and 0·025 g/cm(2) (0·001; 0·049), respectively, than those with levels 2·7-5·6 pmol/l. Participants with normocalcaemic and secondary hyperparathyroidism also had lower total hip BMD than those with levels 2·7-5·6 pmol/l, and CaMos prevalences of normocalcaemic, primary and secondary hyperparathyroidism were 3·3%, 1·4% and 5·2%, respectively. CONCLUSION: We found reduced BMD in participants with accepted hyperparathyroid states but also a notable proportion of other participants that might benefit from having lower PTH levels.

Longitudinal changes in calcium and vitamin D intakes and relationship to bone mineral density in a prospective population-based study: the Canadian Multicentre Osteoporosis Study (CaMos).

OBJECTIVES: Our objective was to study changes in calcium and vitamin D intakes over time, and their cross-sectional and longitudinal associations with bone mineral density (BMD). METHODS: We followed 9382 women and men aged ≥25 and 899 aged 16-24, for 10 and 2 years respectively. RESULTS: Calcium and vitamin D intakes increased over time in adults, but decreased in women aged 16-18. The increased intakes in adults were largely attributable to the increased use of calcium and/or vitamin D supplements. Both the percentage of supplement users and average dose among users increased over time. There was nevertheless a high prevalence of calcium and vitamin D intake below the estimated average requirement. At baseline, higher calcium and vitamin D intakes were associated with higher total hip and femoral neck BMD in young men, and cumulatively high levels of calcium and vitamin D intakes over time contributed to better BMD maintenance at lumbar spine and hip sites in adult women. CONCLUSIONS: Although total intakes, particularly of vitamin D, frequently fell below the Institute of Medicine recommendations despite an increase over time in supplement use, we found some positive associations between total calcium and vitamin D intake and bone health.

Predicting fracture using 2D finite element modelling.

A decrease in bone density at the hip or spine has been shown to increase the risk of fracture. A limitation of the bone mineral density (BMD) measurement is that it provides only a measure of a bone sample's average density when projected onto a 2D surface. Effectively, what determines bone fracture is whether an applied load exceeds ultimate strength, with both bone tissue material properties (can be approximated through bone density), and geometry playing a role. The goal of this project was to use bone geometry and BMD obtained from radiographs and DXA measurements respectively to estimate fracture risk, using a two-dimensional finite element model (FEM) of the sagittal plane of lumbar vertebrae. The Canadian Multicentre Osteoporosis Study (CaMos) data was used for this study. There were 4194 men and women over the age of 50 years, with 786 having fractures. Each subject had BMD testing and radiographs of their lumbar vertebrae. A single two dimensional FEM of the first to fourth lumbar vertebra was automatically generated for each subject. Bone tissue stiffness was assigned based on the BMD of the individual vertebrae, and adjusted for patient age. Axial compression boundary conditions were applied with a force proportional to body mass. The resulting overall strain from the applied force was found. Men and women were analyzed separately. At baseline, the sensitivity of BMD to predict fragility fractures in women and men was 3.77% and 0.86%, while the sensitivity of FEM to predict fragility fractures for women and men was 10.8% and 11.3%. The FEM ROC curve demonstrated better performance compared to BMD. The relative risk of being considered at high fracture risk using FEM at baseline, was a better predictor of 5 year incident fragility fracture risk compared to BMD.

Fracture prediction and calibration of a Canadian FRAX® tool: a population-based report from CaMos.

UNLABELLED: A new Canadian WHO fracture risk assessment (FRAX®) tool to predict 10-year fracture probability was compared with observed 10-year fracture outcomes in a large Canadian population-based study (CaMos). The Canadian FRAX tool showed good calibration and discrimination for both hip and major osteoporotic fractures. INTRODUCTION: The purpose of this study was to validate a new Canadian WHO fracture risk assessment (FRAX®) tool in a prospective, population-based cohort, the Canadian Multicentre Osteoporosis Study (CaMos). METHODS: A FRAX tool calibrated to the Canadian population was developed by the WHO Collaborating Centre for Metabolic Bone Diseases using national hip fracture and mortality data. Ten-year FRAX probabilities with and without bone mineral density (BMD) were derived for CaMos women (N = 4,778) and men (N = 1,919) and compared with observed fracture outcomes to 10 years (Kaplan-Meier method). Cox proportional hazard models were used to investigate the contribution of individual FRAX variables. RESULTS: Mean overall 10-year FRAX probability with BMD for major osteoporotic fractures was not significantly different from the observed value in men [predicted 5.4% vs. observed 6.4% (95%CI 5.2-7.5%)] and only slightly lower in women [predicted 10.8% vs. observed 12.0% (95%CI 11.0-12.9%)]. FRAX was well calibrated for hip fracture assessment in women [predicted 2.7% vs. observed 2.7% (95%CI 2.2-3.2%)] but underestimated risk in men [predicted 1.3% vs. observed 2.4% (95%CI 1.7-3.1%)]. FRAX with BMD showed better fracture discrimination than FRAX without BMD or BMD alone. Age, body mass index, prior fragility fracture and femoral neck BMD were significant independent predictors of major osteoporotic fractures; sex, age, prior fragility fracture and femoral neck BMD were significant independent predictors of hip fractures. CONCLUSION: The Canadian FRAX tool provides predictions consistent with observed fracture rates in Canadian women and men, thereby providing a valuable tool for Canadian clinicians assessing patients at risk of fracture.

Fragility fractures and the osteoporosis care gap in women: the Canadian Multicentre Osteoporosis Study.

UNLABELLED: Canadian women over 50 years old were studied over a 10-year period to see if those who sustained a fracture (caused by minimal trauma) were receiving the recommended osteoporosis therapy. We found that approximately half of these women were not being treated, indicating a significant care gap in osteoporosis treatment. INTRODUCTION: Prevalent fragility fracture strongly predicts future fracture. Previous studies have indicated that women with fragility fractures are not receiving the indicated treatment. We aimed to describe post fracture care in Canadian women using a large, population-based prospective cohort that began in 1995-1997. METHODS: We followed 5,566 women over 50 years of age from across Canada over a period of 10 years in the Canadian Multicentre Osteoporosis Study. Information on medication use and incident clinical fragility fractures was obtained during a yearly questionnaire or interview and fractures were confirmed by radiographic/medical reports. RESULTS: Over the 10-year study period, 42-56% of women with yearly incident clinical fragility fractures were not treated with an osteoporosis medication. During year 1 of the study, 22% of the women who had experienced a fragility fracture were on treatment with a bisphosphonate and 26% were on hormone therapy (HT). We were not able to differentiate HT use for menopause symptoms vs osteoporosis. Use of bisphosphonate therapy increased over time; odds ratio (OR) for use at year 10 compared to use at year 1 was 3.65 (95% confidence interval (CI) 1.83-7.26). In contrast, HT use declined, with an OR of 0.07 (95%CI 0.02-0.24) at year 10 compared to year 1 of the study. CONCLUSION: In a large population-based cohort study, we found a therapeutic care gap in women with osteoporosis and fragility fractures. Although bisphosphonate therapy usage improved over time, a substantial gap remains.

Risk factors for low BMD in healthy men age 50 years or older: a systematic review.

SUMMARY: In this systematic review, we summarize risk factors for low bone mineral density and bone loss in healthy men age 50 years or older. Consistent risk factors were: age, smoking, low weight, physical/functional limitations, and previous fracture. Data specific to men has clinical and policy implications. INTRODUCTION: Osteoporosis is a significant health care problem in men as well as women, yet the majority of evidence on diagnosis and management of osteoporosis is focused on postmenopausal women. The objective of this systematic review is to examine risk factors for low bone mineral density (BMD) and bone loss in healthy men age 50 years or older. MATERIALS AND METHODS: A systematic search for observational studies was conducted in MEDLINE, Cochrane Database of Systematic Reviews, DARE, CENTRAL, CINAHL and Embase, Health STAR. The three main search concepts were bone density, densitometry, and risk factors. Trained reviewers assessed articles using a priori criteria. RESULTS: Of 642 screened abstracts, 299 articles required a full review, and 25 remained in the final assessment. Consistent risk factors for low BMD/bone loss were: advancing age, smoking, and low weight/weight loss. Although less evidence was available, physical/functional limitations and prevalent fracture (after age 50) were also associated with low BMD/bone loss. The evidence was inconsistent or weak for physical activity, alcohol consumption, calcium intake, muscle strength, family history of fracture/osteoporosis, and height/height loss. CONCLUSION: In this systematic review, we identified several risk factors for low BMD/bone loss in men that are measurable in primary practice.

Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors.

Second- and third-generation three-plasmid vector systems, termed FELIX, were constructed from feline immunodeficiency virus (FIV). To enhance vector production, the weak FIV long terminal repeat promoter was replaced with the human cytomegalovirus enhancer/promoter. To construct a minimal system in which Gag-Pol was the only viral protein present, the cytoplasmic transport element was used in place of the FIV Rev-RRE system to facilitate nuclear export of Gag-Pol and the transfer vector. Unconcentrated vector titers routinely exceeded 1 x 10(6) IU/mL for most constructs tested. Second- and optimized third-generation vectors were capable of efficiently infecting G1/S- and G2/M-arrested cells. FIV-based FELIX vectors transduced human dendritic cells, hepatocytes, and aortic smooth muscle with efficiencies similar to that of a control 3T3 cell line. All three of these primary cell types were transducible by both the second- and third-generation FELIX vectors, demonstrating that FIV Gag-Pol alone contains the determinants necessary for transduction of primary cells. In cross-packaging tests, we observed that HIV Gag-Pol does not substantially package FIV vectors; consequently, use of such vectors in human immunodeficiency virus-infected cells should not lead to efficient mobilization of the inserted gene. Thus, this FIV-based vector system offers high efficiency and stable delivery of genes to numerous nondividing and primary cell types, opening new avenues for biological inquiry into normal human cells.

Diimidazo[1,2-c:4',5'-e]pyrimidines: adenosine agonist activity demonstrated by microphysiometry.

Silicon-based microphysiometry, measuring extracellular acidification rate of cells in culture, demonstrated that a series of diimidazo[1,2-c:4',5'-e]pyrimidines were agonists at the human adenosine A1 receptor. 5-amino-7,8-dihydro-3-ribofuranose-8-(R)-(phenyl)-3H-diimidazo [1,2-c:4',5'-e]pyrimidine (2a) had an EC50 of 100 microM and reached 90% of the Emax produced by R-PIA.

Antisense-mediated inhibition of parathyroid hormone-related peptide production in a keratinocyte cell line impedes differentiation.

We have used antisense RNA technology to inhibit endogenous PTH-related peptide (PTHRP) production in an established human keratinocyte cell line, HPK1A, to assess the role of PTHRP as a modulator of cell differentiation. Initially to determine the specificity of any alterations in cell function that might be observed, HPK1A cells and Rat-2 fibroblasts (which do not synthesize PTHRP) were both infected with the same retrovirus (pYN) containing antisense PTHRP. In contrast to antisense-infected HPK1A cells (HPK1A-AS), which show accelerated growth indices when endogenous PTHRP production is blocked, antisense-infected Rat-2 cells (Rat-2-AS) displayed no increase in cell proliferation. Consequently, this alteration in HPK1A cell function appeared to be specific to the inhibition of PTHRP production. In HPK1A-AS cells, no PTHRP transcript was observed in cytoplasmic RNA, and none was sequestered in a nuclear RNA preparation. Therefore, hybridization with the antisense strand appears to destabilize PTHRP mRNA, leading to rapid disappearance of the sense-antisense heteroduplex. We then examined the effect of PTHRP inhibition on keratinocyte differentiation using three indices. PTHRP inhibition in HPK1A-AS cells resulted in reduced high mol wt keratin production, as assessed by immunocytochemistry. Expression of mRNA encoding transglutaminase and involucrin was decreased in HPK1A-AS cells compared to that in control cells under conditions of high ambient calcium. Involucrin protein levels were also diminished in HPK1A-AS cells in parallel with the reduced levels of involucrin gene expression. These data, therefore, show that interference with PTHRP production inhibits expression of maturation-specific keratinocyte indices and indicate that endogenous PTHRP acts to enhance differentiation in this keratinocyte model.

Parathyroid hormone-related peptide.

Parathyroid hormone-related peptide (PTHRP) was discovered through its ability to cause parathyroid hormone (PTH)-like effects in malignancy-associated hypercalcemia (MAH). It shares limited amino-terminal sequence homology with PTH and can thus interact with PTH receptors. It was originally isolated, cloned, and sequenced from tumors of patients with MAH. The human PTHRP gene is complex and can generate 3 peptide isoforms of 139, 141, and 173 amino acids. The gene encoding PTHRP in other species is somewhat simpler and generally results in only a single isoform; however, there is marked interspecies amino acid sequence homology in the protein product up to residue 111. PTH and PTHRP genes have similar structural organizations and it is believed that these 2 genes have a common ancestral origin and may have arisen through an ancient gene duplication. There are now several region specific radioimmunoassays, as well as immunoradiometric assays, which have been developed and which have all demonstrated elevated circulating levels of PTHRP in patients with MAH. PTHRP is expressed in a wide variety of tissues and, in addition to its classical PTH-like activities, it may have a local or systemic role in calcium regulation in the fetus and perhaps in the neonate. It may also act locally as a smooth muscle relaxant and a vasorelaxant. Its local role in some fetal tissues and in keratinocytes may take the form of a regulator of cell growth and/or differentiation. Thus when antisense RNA technology was used to inhibit endogenous PTHRP production in an established human keratinocyte cell line, PTHRP was found to inhibit growth and enhance differentiation. Irrespective of its precise normal role however, PTHRP provides a potential therapeutic target in MAH and, with improved methods of detection, may also be useful as a tumor marker.

Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide.

We have used antisense RNA technology to inhibit endogenous parathyroid hormone-related peptide (PTHRP) production in the established human keratinocyte cell line, HPK1A, in order to assess the role of PTHRP as a potential modulator of cell growth. Rat PTHRP cDNA was cloned into the replication defective retroviral vector pYN in an antisense orientation and a stable cell line (HPK1A-AS) was generated after infection by amphotropic virus and selection by the neomycin derivative, G418. Expression of the transfected antisense sequence was confirmed with an RNA sense probe for PTHRP. The effect of the retrovirally mediated gene transfer on the endogenous PTHRP transcript was examined with an RNA antisense probe which demonstrated an absence of the endogenous transcript in HPK1A-AS cells. A 1.6-kilobase transcript was, however, present in equivalent quantities in both uninfected HPK1A and pYN-infected (HPK1A-pYN) cells. Immunocytochemistry and assessment of PTHRP secretion into the medium using an NH2-terminal radioimmunoassay and a UMR 106 adenylate cyclase bioassay confirmed the absence of PTHRP in HPK1A-AS cells. Examination of the inhibition of PTHRP production on cell growth demonstrated a reduction in doubling time and an increase in [3H]thymidine incorporation. Cell cycle analysis showed an increase in the proportion of the cell population in the S phase (relative to G0/G1) in HPK1A-AS cells compared to HPK1A or HPK1A-pYN cells. These data, therefore, indicate that endogenous PTHRP acts as an effective inhibitor of cell growth in this keratinocyte model and that this action occurs, at least in part, by diminishing entry into the S phase of the cell cycle. Furthermore, the antisense RNA method is a potent one to evaluate the cellular actions of PTHRP.

Synthesis and characterization of extended and deleted recombinant analogues of parathyroid hormone-(1-84): correlation of peptide structure with function.

Recombinant analogues of human parathyroid hormone [hPTH-(1-84)] were expressed in Escherichia coli harboring plasmids containing synthetic genes under the control of the lac promoter. The level of expression of the gene encoding the truncated analogue, hPTH-(3-84), was greater than that of the gene encoding full-length hPTH-(1-84) but less than that of the gene encoding proparathyroid hormone (hProPTH). This may be due in part to the relative efficiency of translation of the mRNA as suggested by secondary structure analysis and in part because of enhanced stability of the extended peptide. Formylmethionyl derivatives of hProPTH and of hPTH-(3-84) and underivatized hPTH-(3-84) were purified by HPLC, and their identity was confirmed by NH2-terminal sequencing and amino acid analysis. The bioactivity of these recombinant peptides was then tested in skeletal and renal adenylate cyclase assays in vitro and in assays examining effects on plasma and urine calcium and phosphate levels and on urine cyclic AMP levels in vivo. The NH2-terminally extended analogue fMet-hProPTH displayed 10% of the in vitro activity of hPTH-(1-84) and was a partial agonist in vivo. The peptides hPTH-(3-84) and fMet-hPTH-(3-84) were inert in vitro and were very weak in vitro antagonists when compared to the NH2-terminal analogue bovine [Nle8,18Tyr34]PTH-(3-34)-NH2. In vivo, hPTH-(3-84) and the bPTH-(3-34) analogue, when assayed at a 10:1 molar ratio relative to bPTH-(1-84), were each inert, and neither demonstrated antagonist activity at these concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)