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Leonard J Deftos

Publications and source records attributed to Leonard J Deftos.

At least 19 recordsLinked to original sources
PubMed2006

The bisphosphonate olpadronate inhibits skeletal prostate cancer progression in a green fluorescent protein nude mouse model.

PURPOSE: Metastatic bone disease is one of the major causes of morbidity and mortality in prostate cancer patients. Bisphosphonates are currently used to inhibit bone resorption and reduce tumor-induced skeletal complications. More effective bisphosphonates would enhance their clinical value. EXPERIMENTAL DESIGN: We tested several bisphosphonates in a green fluorescent protein (GFP)-expressing human prostate cancer nude mouse model. The in vivo effects of four bisphosphonates, including pamidronate, etidronic acid, and olpadronate, on bone tumor burden in mice intratibially inoculated with PC-3-GFP human prostate cancer cells were visualized by whole-body fluorescence imaging and X-ray. RESULTS: The PC-3-GFP cells produced extensive bone lesions when injected into the tibia of immunocompromised mice. The skeletal progression of the PC-3-GFP cell growth was monitored by GFP fluorescence and the bone destruction was evaluated by X-ray. We showed that 3,3-dimethylaminopropane-1-hydroxy-1,1-diphosphonic acid (olpadronate) was the most effective bisphosphonate treatment in reducing tumor burden as assessed by GFP imaging and radiography. The GFP tumor area and X-ray score significantly correlated. Reduced tumor growth in the bone was accompanied by reduced serum calcium, parathyroid hormone-related protein, and osteoprotegerin. CONCLUSIONS: The serum calcium, parathyroid hormone-related protein, and osteoprotegerin levels were significantly correlated with GFP area and X-ray scores. Treatment with olpadronate reduced tumor growth in the bone measured by GFP and X-ray imaging procedures. Imaging of GFP expression enables monitoring of tumor growth in the bone and the GFP results complement the X-ray assessment of bone disease. The data in this report suggest that olpadronate has potential as an effective inhibitor of the skeletal progression of clinical prostate cancer.

Animals↗
PubMed2006

Identification of DU 145 prostate cancer cell proteins that bind to the carboxy-terminal peptide of human PTHrP in vitro.

Peptides spanning the range of human parathyroid hormone-related protein (PTHrP) have been shown to bind heat shock protein-70 expressed on the surface of cancer cells with cytoprotective consequences in vitro. The present study focused on identification of intracellular proteins that interact with the carboxy-terminal peptide of human PTHrP. Using affinity chromatography, we applied extracts of DU 145 prostate cancer cells over PTHrP (140-173)-Sepharose and eluted with 8 M urea. After concentration and electrophoresis, protein bands were excised and subjected to mass spectroscopy analyses. Proteins identified included those associated with protection from oxidative stress, DNA repair, protection from apoptosis, and proteins involved in membrane trafficking and cytoskeletal rearrangement. These novel protein-protein interactions further support the hypothesis that the carboxy-terminus of PTHrP plays a role in cell survival.

Biotinylation↗
PubMed2006

Sex-specific survival advantage with parathyroid hormone-related protein in non-small cell lung carcinoma patients.

PURPOSE: Parathyroid hormone-related protein (PTHrP) is commonly expressed in non-small cell lung carcinomas (NSCLC). Expression of the protein could have implications for progression of the disease because it regulates cancer cell growth, apoptosis, and angiogenesis. However, its relationship with survival has not been evaluated in a large-scale investigation. EXPERIMENTAL DESIGN: PTHrP expression was assessed in paraffin-embedded tumor samples from 407 patients with NSCLC by immunohistochemistry. A pathologist unaware of the clinical history classified specimens as PTHrP positive or PTHrP negative. The log-rank test was used to compare survivals of PTHrP-positive and PTHrP-negative groups, and Cox regression was used to adjust for additional covariates. RESULTS: Median survival was 55 versus 22 months (P < 0.001) in female patients with and without tumor PTHrP, respectively. Male survival was 38 months independent of PTHrP status. Stage, histology, age, and smoking history were also associated with increased longevity. PTHrP remained a significant predictor of survival for female patients after controlling for stage, histology, and age. CONCLUSIONS: In this study, PTHrP expression was associated with a survival advantage in female patients. Additional investigations must be done to ascertain whether the result is reproducible and independent of potential confounding covariates. Sex-dependent effects of PTHrP in lung cancer would open new avenues of research into the role of sex in cancer progression.

Adenocarcinoma↗
PubMed2005

A carboxyl leucine-rich region of parathyroid hormone-related protein is critical for nuclear export.

PTHrP is an oncofetal protein with distinct proliferative and antiapoptotic roles that are affected by nucleocytoplasmic shuttling. The protein's nuclear export is sensitive to leptomycin B, consistent with a chromosome region maintenance protein 1-dependent pathway. We determined that the 109-139 region of PTHrP was involved in its nuclear export by demonstrating that a C-terminal truncation mutant, residues 1-108, exports at a reduced rate, compared with the wild-type 139 amino acid isoform. We searched for potential nuclear export sequences within the 109-139 region, which is leucine rich. Comparisons with established nuclear export sequences identified a putative consensus signal at residues 126-136. Deletion of this region resulted in nuclear export characteristics that closely matched those of the C-terminal truncation mutant. Confocal microscopic analyses of transfected 293, COS-1, and HeLa cells showed that steady-state nuclear levels of the truncated and deletion mutants were significantly greater than levels of wild-type PTHrP and were unaffected by leptomycin B, unlike the wild-type protein. In addition, both mutants demonstrated greatly reduced nuclear export with assays using nuclear preparations and intact cells. Based on these results, we conclude that the 126-136 amino acid sequence closely approximates the structure of a chromosome region maintenance protein 1-dependent leucine-rich nuclear export signal and is critical for nuclear export of PTHrP.

Active Transport, Cell Nucleus↗
PubMed2005

41Ca and accelerator mass spectrometry to monitor calcium metabolism in end stage renal disease patients.

BACKGROUND: Monitoring bone resorption with measurements of bone density and biochemical markers is indirect. We hypothesized that bone resorption can be studied directly by serial measurements of the ratio (41)Ca/Ca in serum after in vivo labeling of calcium pools with (41)Ca. We report the preparation of an intravenous (41)Ca dose suitable for humans, an analytical method for determining (41)Ca/Ca isotope ratios in biological samples, and studies in human volunteers. METHODS: (41)Ca was formulated and aliquoted into individual vials, and to the extent possible, the (41)Ca doses were tested according to US Pharmacopeia (USP) guidelines. A 10 nCi dose of (41)Ca was administered intravenously to 4 end stage renal disease (ESRD) patients on hemodialysis and 4 healthy control individuals. Distribution kinetics were determined over 168 days. Calcium was isolated with 3 precipitation steps and a cation-exchange column, and (41)Ca/Ca ratios in serum were then measured by accelerator mass spectrometry. RESULTS: The dosing solution was chemically and radiologically pure, contained <0.1 endotoxin unit/mL, and passed USP sterility tests. Quantification of (41)Ca/Ca ratios was linear from 6 x 10(-14) to 9.1 x 10(-10). The run-to-run imprecision (as CV) of the method was 4% at 4.6 x 10(-11) and 6% at 9.1 x 10(-10). The area under the curve of (41)Ca in the central compartment vs time was significantly less for ESRD patients than for controls (P < 0.005). CONCLUSIONS: Isotope ratios spanning 5 orders of magnitude can be measured by accelerator mass spectrometry with excellent precision in the range observed in samples collected from patients who have received 10 nCi of (41)Ca. The (41)Ca at this dose caused no adverse effects in 8 volunteers. This is the first report of the use of (41)Ca to monitor differences in bone turnover between healthy individuals and ESRD patients.

Bone and Bones↗
PubMed2005

Heat shock protein-70 expressed on the surface of cancer cells binds parathyroid hormone-related protein in vitro.

Recent studies have shown that the functions of PTH-related protein (PTHrP) and its derived peptides cannot be attributed solely to PTH/PTHrP receptor binding. The present study focused on the identification of other proteins that might bind PTHrP at the cell surface. Using affinity chromatography, we applied extracts of cell-surface biotinylated proteins from cancer and normal cell lines over Sepharose beads coupled with different PTHrP-derived peptides. Elution with the corresponding free peptide revealed a major protein of about 70 kDa that was present in all of the PTHrP peptide eluates from cancer cell extracts but not from normal breast cell extracts. Mass spectroscopy analysis and immunoblotting identified this PTHrP-binding protein as heat shock protein-70 (HSP70). Using a recently published algorithm that predicts HSP70 binding sites within proteins, we found that all four PTHrP peptides used in these studies contain amino acid motifs with high probabilities for HSP70 binding in vivo. Cell culture studies in the presence of a polyclonal anti-HSP70 antibody demonstrated increased PTHrP secretion, decreased total cellular protein, and differentially regulated proliferation. Taken together, these studies demonstrate a novel and biologically relevant interaction between cell surface-expressed HSP70 and PTHrP in cancer.

Adenocarcinoma↗
PubMed2005

GSK3 and PKB/Akt are associated with integrin-mediated regulation of PTHrP, IL-6 and IL-8 expression in FG pancreatic cancer cells.

We have demonstrated recently that PTHrP is upregulated in pancreatic adenocarcinoma and that the ECM exerts regulatory control, at least in part, over PTHrP expression. In our present study, we examined the potential signaling interactions between these 2 pathways. Our results demonstrate that, under serum-free conditions, adhesion of FG pancreatic adenocarcinoma cells on Fn is mediated by the alpha5beta1 integrin, whereas adhesion to Type I collagen is mediated by the alpha2beta1 integrin. alpha5beta1 integrin-mediated adhesion to Fn results in a phenotype that includes a reduction in cell proliferation, increased E-cadherin localization in cell-cell contacts, increased beta-catenin localization throughout the cell, inhibition of haptokinetic cell migration, and increased expression of PTHrP, IL-6 and IL-8 relative to alpha2beta1 integrin-mediated adhesion on Type I collagen. A phosphoprotein immunoblotting screen of FG pancreatic cancer cells grown on either Fn or Type I collagen indicates that GSK3 and PKB/Akt are differentially phosphorylated on these 2 substrates. These results implicate GSK3 and PKB/Akt in the integrin-mediated regulation of PTHrP, IL-6 and IL-8 in pancreatic cancer.

Adenocarcinoma↗
PubMed2005

Direct evidence that PTHrP expression promotes prostate cancer progression in bone.

Parathyroid hormone-related protein (PTHrP) is an oncoprotein that is expressed in many malignancies as well as normal tissues. At essentially every site of expression, PTHrP regulates cell growth and proliferation. We and other investigators have previously reported that PTHrP is widely expressed by prostate cancer. For this tumor, there are substantial in vitro and correlative data that PTHrP expression regulates the progression of the tumor, especially in bone, but little direct data. We studied the effects of PTHrP expression on prostate cancer behavior directly in a mouse model of human prostate cancer cells that were transfected to express different forms of the polypeptide and then injected intraskeletally. Skeletal progression of the prostate cancer cells was evaluated radiologically and by measurement of serum tumor markers. PTHrP transfection converted a non-invasive cell line into one that progressed in the skeleton: Injection of the PTHrP transfected cells resulted in greater tumor progression in bone when compared to non-transfected cells, and this effect was also influenced by non-amino terminal peptides of PTHrP. Serum measurements of PTHrP, IL-6, IL-8, and calcium reflected tumor burden. Our experiments provide direct in vivo evidence that PTHrP expression results in the skeletal progression of prostate cancer cells.

Animals↗
PubMed2005

Type I collagen and divalent cation shifts disrupt cell-cell adhesion, increase migration, and decrease PTHrP, IL-6, and IL-8 expression in pancreatic cancer cells.

BACKGROUND: We have shown in FG pancreatic cancer cells that alpha2beta1 integrin-mediated type I collagen adhesion decreases parathyroid hormone-related protein (PTHrP), interleukin-6 (IL-6), and interleukin-8 (IL-8) expression, decreases the localization of E-cadherin and beta-catenin in cell-cell contacts, increases cell migration, and increases glycogen synthase kinase 3 (GSK3) and protein kinase B (PKB/Akt) phosphorylation states relative to alpha5beta1 integrin-mediated fibronectin (Fn) adhesion. AIM OF THE STUDY: To extend our observations in FG cells to other pancreatic cancer cell lines, and to determine whether E-cadherin-mediated cell-cell adhesion and its downstream effectors were functionally involved in the ECM-mediated regulation of PTHrP, IL-6, and IL-8. METHODS: We used standard biochemical techniques to determine ECM-specific differences in E-cadherin and beta-catenin localization, GSK3 and PKB/Akt phosphorylation, haptokinetic cell migration, and cytokine expression in pancreatic cancer cells. We also conducted functional studies using pharmacological inhibitors for GSK3 and PKB/Akt, as well as elevated Mg2+/Ca2+ ratios similar to pancreatic juice, and examined their effects on cytokine expression. RESULTS: Differences in E-cadherin and beta-catenin localization along with GSK3 and PKB/Akt phosphorylation occur in multiple pancreatic cancer cell lines, resulting in differences in ECM-mediated haptokinesis and cytokine expression that are generally consistent with previous observations in FG cells. Our functional studies also suggest that E-cadherin-mediated cell-cell adhesion and downstream effectors are involved in PTHrP, IL-6, and IL-8 expression. CONCLUSIONS: These data indicate that alpha2beta1 integrin-mediated type I collagen adhesion disrupts cell-cell adhesion architecture, resulting in increased migration and decreased PTHrP, IL-6, and IL-8 expression in pancreatic cancer cells.

Adenocarcinoma↗
PubMed2004

Parathyroid hormone-related protein expression is correlated with clinical course in patients with glial tumors.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) expression modulates cell survival in a number of human solid tumors. Although PTHrP is expressed in normal developing and neoplastic central nervous system tissue, clinical data indicating the importance of this protein with respect to local control and/or survival in patients with glial tumors are scarce. METHODS: Using a standard immunoperoxidase technique, the authors examined PTHrP expression in a population of 51 patients with Daumas-Duport Grade II-IV astrocytomas over a 15-year period. Both local control and survival were calculated from the date of definitive irradiation to the last time of known follow-up examination using the actuarial method. PTHrP expression was scored on examination under 40x magnification, with the incidence of cellular staining averaged over 10 high-power fields. The intensity and extent of staining were characterized semiquantitatively using the standard World Health Organization classification criteria. The median follow-up duration was approximately 5.5 years. Multivariate analyses were performed to ascertain the statistical significance of several standard clinicohistopatholgic factors (Karnofsky functional status, age, gender, extent of surgical resection, radiotherapy dose, grade, and PTHrP expression) with respect to local control and survival. P < 0.05 was considered indicative of statistical significance. RESULTS: Patients with high levels of PTHrP expression had significantly lower glial tumor local control rates and corresponding decreases in progression-free and overall actuarial survival after definitive irradiation (P < 0.01). In a Cox 3-variable model, the PTHrP staining score was independent of tumor grade or Karnofsky functional status. It is notable that the strongest predictor of survival was tumor grade (P < 0.001). CONCLUSIONS: PTHrP may be an important adjunct to standard immunopathologic criteria in the determination of glial tumor responses. A number of mechanisms were explored to derive a more mechanistic understanding of these translational results. Subsequent prospective studies involving larger patient populations will be necessary before findings can be translated to clinical practice.

Central Nervous System Neoplasms↗
PubMed2004

Parathyroid hormone-related protein regulates apoptosis in lung cancer cells through protein kinase A.

Parathyroid hormone-related protein (PTHrP)-(1-34) and PTHrP-(140-173) protect lung cancer cells from apoptosis after ultraviolet (UV) irradiation. This study evaluated upstream signaling in PTHrP-mediated alteration of lung cancer cell sensitivity to apoptosis. The two peptides increased cAMP levels in BEN lung cancer cells by 15-35% in a dose-dependent fashion, suggesting signaling through protein kinase A (PKA). In line with this view, the PKA inhibitor H89 abrogated the protective effects of PTHrP-(1-34) and PTHrP-(140-173) against caspase activation and DNA loss. PKA activation by forskolin, 3-isobutyl-1-methylxanthine (IBMX), or 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate attenuated and H89 augmented apoptosis after UV exposure as indicated by caspase-3 activation, cell DNA loss, and morphological criteria. Studies with IBMX and varying doses of forskolin indicated that small increases in cAMP, on the order of those generated by IBMX alone and the PTHrP peptides, were sufficient to protect lung cancer cells from apoptosis. In summary, PTHrP-(1-34) and PTHrP-(140-173) stimulate PKA in lung carcinoma cells and protect cells against UV-induced caspase-3 activation and DNA fragmentation. PKA activation by other means also induces resistance to apoptosis, and the protective effect of the PTHrP peptide is blocked by PKA inhibition. Thus PKA appears to have a role in the regulatory effects of PTHrP on lung cancer cell survival.

Caspases↗
PubMed2004

The extracellular matrix differentially regulates the expression of PTHrP and the PTH/PTHrP receptor in FG pancreatic cancer cells.

OBJECTIVES: Previous studies by our laboratory have demonstrated that parathyroid hormone-related protein (PTHrP) and its receptor (PTH/PTHrP receptor) are commonly expressed in pancreatic cancer and suggest their participation in the progression of this devastating disease. It has also been demonstrated that one of the major hallmarks of pancreatic adenocarcinoma is an increased production of the extracellular matrix (ECM), a critical regulator of diverse cellular processes such as differentiation, proliferation, and angiogenesis. The present study focused on the relationship between the PTHrP and ECM axes in the pathobiology of pancreatic cancer. METHOD AND RESULTS: Using the FG pancreatic adenocarcinoma cell line, we demonstrate a significant inverse correlation between FG cell proliferation and PTHrP expression that depended on the ECM protein on which the cells were cultured (P < 0.05). Generally, ECM proteins that promoted the strongest proliferation, including type I collagen, type IV collagen, and laminin, resulted in decreased expression of PTHrP. Conversely, ECM proteins that promoted the weakest proliferation, including fibronectin, vitronectin, and BSA, resulted in increased expression of PTHrP. A similar trend was found between FG cell proliferation and the PTH/PTHrP receptor expression, with Pearson correlation coefficients of 0.480 (mRNA) and -0.591 (protein). CONCLUSION: These observations demonstrate a unique functional relationship between the ECM and PTHrP axes and have important implications for our understanding of the complex mechanisms responsible for the progression of pancreatic cancer and its metastases.

Adenocarcinoma↗
PubMed2003

Amino-terminal and midmolecule parathyroid hormone-related protein, phosphatidylcholine, and type II cell proliferation in silica-injured lung.

Acute silica lung injury is marked by alveolar phospholipidosis and type II cell proliferation. Parathyroid hormone-related protein (PTHrP) 1-34 could have a regulatory role in this process because it stimulates phosphatidylcholine secretion and inhibits type II cell growth. Other regions of the PTHrP molecule may have biological activity and can also exert pulmonary effects. This study examined the temporal pattern for expression of several regions of PTHrP after silica lung injury and evaluated the effects of changes in expression on cell proliferation and lung phospholipids. Expression of all PTHrP regions fell at 4 days after injury. Reversing the decline in PTHrP 1-34 or PTHrP 67-86 with one intratracheal dose and four daily subcutaneous doses of PTHrP 1-34 or PTHrP 67-86 stimulated bronchoalveolar lavage disaturated phosphatidylcholine (DSPC) levels. Cell culture studies indicate that the peptides exerted direct effects on DSPC secretion by type II cells. Neither peptide affected type II cell proliferation with this dosing regimen, but addition of an additional intratracheal dose resulted in significant inhibition of growth, consistent with previous effects of PTHrP 1-34 in hyperoxic lung injury. These studies establish a regulatory role for PTHrP 1-34 and PTHrP 67-86 in DSPC metabolism and type II cell proliferation in silica injury. Growth inhibitory effects of PTHrP could interact with phospholipid stimulation by affecting type II cell numbers. Further studies are needed to explore the complex interactions of PTHrP-derived peptides and the type II cell response at various stages of silica lung injury.

Animals↗
PubMed2003

Parathyroid hormone-related protein ameliorates death receptor-mediated apoptosis in lung cancer cells.

Parathyroid hormone-related protein (PTHrP) is expressed in more advanced, aggressive tumors and may play an active role in cancer progression. This study investigated the effects of PTHrP on apoptosis after UV irradiation, Fas ligation, or staurosporine treatment in BEN human squamous lung carcinoma cells. Cells at 70% confluency were treated for 24 h with 100 nM PTHrP-(1-34), PTHrP-(38-64), PTHrP-(67-86), PTHrP-(107-139), or PTHrP-(140-173) in media with serum, exposed for 30 min to UV-B radiation (0.9 mJ/cm2), and maintained for another 24 h. Caspase-3, caspase-8, and caspase-9 activities increased fivefold. Pretreatment with PTHrP-(1-34) and PTHrP-(140-173) ameliorated apoptosis after UV irradiation, as indicated by reduced caspase activities, increased cell protein, decreased nuclear condensation, and increased clonal survival. Other peptides had no effect on measures of apoptosis. PTHrP-(140-173) also reduced caspase activities after Fas ligation by activating antibody, but neither peptide had effects on caspase-3 or caspase-9 activity after 1 microM staurosporine. These data indicate that PTHrP-(1-34) and PTHrP-(140-173) protect against death receptor-induced apoptosis in BEN lung cancer cells but are ineffective against mitochondrial pathways. PTHrP contributes to lung cancer cell survival in culture and could promote cancer progression in vivo. The mechanism for the protective effect against apoptosis remains to be determined.

Apoptosis↗
PubMed2003

Proapoptotic effects of parathyroid hormone-related protein in type II pneumocytes.

Parathyroid hormone-related protein (PTHrP) promotes or suppresses apoptosis in various settings depending on cell type and context. PTHrP 1-34 and PTHrP 67-86 are type II cell growth factors with effects on pneumocyte growth and surfactant secretion. This study investigated the effects of 24 h pretreatment with these two peptides on rat type II cell apoptosis after 0.3 J/cm2 ultraviolet-B irradiation. Adherent cells decreased in number by 15 +/- 5% and nonadherent cells increased > 5-fold 24 h after ultraviolet irradiation. Cell loss was due predominantly to apoptosis, based on ethidium bromide exclusion, nuclear condensation, and caspase 3 activity. Nuclear condensation increased from 15.6 +/- 2.2% of irradiated cells with no treatment to 25.6 +/- 4.9 and 22.9 +/- 1.8% of cells in ultraviolet/PTHrP 1-34 and ultraviolet/PTHrP 67-86 groups, respectively (P < 0.01), along with a 60% increase in caspase 3 activity. Effects on apoptosis were unaffected by the presence or absence of serum, but were ameliorated by growth to confluence or adherence to fibronectin. PTHrP 1-34 and PTHrP 67-86 augmented inositol phosphate levels, but had minimal effects on cAMP. Thus, PTHrP 1-34 and PTHrP 67-86 sensitize type II cells to apoptosis, possibly by a phospholipase C-dependent mechanism. The effects appear to be regulated by cell-matrix and cell-cell interactions.

Animals↗