Cellular carbon stress is a mediator of obesity-associated osteoarthritis development.

OBJECTIVE: 'Carbon stress' is a newly found mechanism that links obesity and dysregulated metabolism. It is defined as the cellular accumulation of metabolites during obesity post-translationally modifying metabolic proteins and decreasing their enzymatic activity. The objective of this study was to investigate if 'carbon stress' also occurs in cartilage and contributes to obesity associated OA development. METHODS: We histologically evaluated for OA pathology in wild-type (WT) and hyperphagic mice (Pomc-neuron specific enhancer one deficient, PomcΔ1) that were subjected to standard chow (Chow, n = 6 for both genotypes) or high-fat feeding (HFD, n = 7 for both genotypes). Joints were stained and quantified for 'carbon stress' markers, including succinyl-lysine (SCK), malonyl-lysine (MAK), and acetyl-lysine (ACK). Lastly, we used a mouse model with deletion of Sirt5 (n = 7), which is an enzyme that removes SCK and MAK, to test if changing the abundance of 'carbon stress' would affect OA pathogenesis. RESULTS: Both HFD and Pomc deficiency associated obesity induced cartilage degeneration as well as greater abundance of SCK and MAK in the cartilage. PomcΔ1-HFD mice did not have exacerbated OA pathology as compared to PomcΔ1-Chow mice. ACK was mildly increased in the obese groups comparing to WT-Chow. Sirt5-/- mice developed early-OA like phenotype at 40 weeks of age as characterized by cartilage fibrillation and more hypertrophic chondrocytes. Cartilage from Sirt5-/- mice also had increased SCK and MAK, while ACK remained unchanged comparing to WT mice. CONCLUSION: Our data suggests that carbon stress also occurs in cartilage tissue during obesity and can potentially contribute to obesity-associated OA.

C-terminal telopeptide of type I collagen, osteocalcin, alkaline phosphatase, and parathyroid hormone in healthy and hospitalized foals.

Hypocalcemia is a common finding in critically ill equine patients. Parathyroid hormone (PTH) helps to maintain calcium homeostasis in hypocalcemic patients by promoting renal calcium reabsorption and bone resorption. Increased serum PTH concentrations have been reported in critically ill people and animals, including horses and foals. It is unknown whether increased secretion of PTH is associated with markers of bone turnover in hospitalized foals. The goals of this study were to measure markers of bone resorption (C-terminal telopeptide of type I collagen [CTX-I]) and bone formation (osteocalcin [OCN]; alkaline phosphatase [ALP]) and to determine their association with PTH concentrations, disease severity, and mortality in hospitalized foals. This prospective, multicenter, cross-sectional study was conducted on 75 newborn foals ≤3 d old divided into hospitalized (n = 65; 41 septic; 24 sick nonseptic) and healthy (n = 10) groups. Blood samples were collected on admission to measure serum CTX-I, OCN, and PTH concentrations and ALP activity. Data were analyzed by nonparametric methods and univariate logistic regression. Serum CTX-I and PTH concentrations were significantly higher, whereas OCN concentrations were lower, in septic compared with healthy foals (P < 0.05). Serum ALP activity was not different between groups; however, it was lower in hospitalized and septic foals with low OCN concentrations (P < 0.05). In hospitalized foals, PTH concentrations were positively correlated with CTX-I concentrations and inversely associated with ALP activity (P < 0.05). High CTX-I and low OCN concentrations were associated with disease severity (P < 0.05). Hospitalized nonsurviving foals had significantly lower OCN concentrations compared with survivors (P < 0.05), but CTX-I concentrations were not associated with survival. Hospitalized foals with PTH concentrations >12.4 pmol/L were more likely to die (OR = 1.5; 95% CI = 1.1-4.16; P < 0.05). Elevated PTH and CTX-I together with reduced OCN concentrations and ALP activity in sick foals indicates that bone resorption is increased during critical illness, which may be a compensatory mechanism to correct hypocalcemia or reflect a response to systemic inflammation and metabolic imbalances. Bone resorption could negatively impact skeletal development in the growing foal. Low OCN and high PTH concentrations were predictors of nonsurvival in hospitalized foals.

Feline Mammary Cancer.

Feline mammary carcinoma (FMC) is similar to human breast cancer in the late age of onset, incidence, histopathologic features, biological behavior, and pattern of metastasis. Therefore, FMC has been proposed as a relevant model for aggressive human breast cancer. The goals of this study were to develop a nude mouse model of FMC tumor growth and metastasis and to measure the expression of genes responsible for lymphangiogenesis, angiogenesis, tumor progression, and lymph node metastasis in FMC tissues and cell lines. Two primary FMC tissues were injected subcutaneously, and 6 FMC cell lines were injected into 3 sites (subcutaneous, intratibial, and intracardiac) in nude mice. Tumors and metastases were monitored using bioluminescent imaging and characterized by gross necropsy, radiology, and histopathology. Molecular characterization of invasion and metastasis genes in FMC was conducted using quantitative real-time reverse transcription polymerase chain reaction in 6 primary FMC tissues, 2 subcutaneous FMC xenografts, and 6 FMC cell lines. The histologic appearance of the subcutaneous xenografts resembled the primary tumors. No metastasis was evident following subcutaneous injection of tumor tissues and cell lines, whereas lung, brain, liver, kidney, eye, and bone metastases were confirmed following intratibial and intracardiac injection of FMC cell lines. Finally, 15 genes were differentially expressed in the FMC tissues and cell lines. The highly expressed genes in all samples were PDGFA, PDGFB, PDGFC, FGF2, EGFR, ERBB2, ERBB3, VEGFD, VEGFR3, and MYOF. Three genes ( PDGFD, ANGPT2, and VEGFC) were confirmed to be of stromal origin. This investigation demonstrated the usefulness of nude mouse models of experimental FMC and identified molecular targets of FMC progression and metastasis.

Animal Models of Bone Metastasis.

Bone is one of the most common sites of cancer metastasis in humans and is a significant source of morbidity and mortality. Bone metastases are considered incurable and result in pain, pathologic fracture, and decreased quality of life. Animal models of skeletal metastases are essential to improve the understanding of the molecular pathways of cancer metastasis and growth in bone and to develop new therapies to inhibit and prevent bone metastases. The ideal animal model should be clinically relevant, reproducible, and representative of human disease. Currently, an ideal model does not exist; however, understanding the strengths and weaknesses of the available models will lead to proper study design and successful cancer research. This review provides an overview of the current in vivo animal models used in the study of skeletal metastases or local tumor invasion into bone and focuses on mammary and prostate cancer, lymphoma, multiple myeloma, head and neck squamous cell carcinoma, and miscellaneous tumors that metastasize to bone.

Combined zoledronic acid and meloxicam reduced bone loss and tumour growth in an orthotopic mouse model of bone-invasive oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is common in cats and humans and invades oral bone. We hypothesized that the cyclooxygenase (COX)-2 inhibitor, meloxicam, with the bisphosphonate, zoledronic acid (ZOL), would inhibit tumour growth, osteolysis and invasion in feline OSCC xenografts in mice. Human and feline OSCC cell lines expressed COX-1 and COX-2 and the SCCF2 cells had increased COX-2 mRNA expression with bone conditioned medium. Luciferase-expressing feline SCCF2Luc cells were injected beneath the perimaxillary gingiva and mice were treated with 0.1 mg kg(-1) ZOL twice weekly, 0.3 mg kg(-1) meloxicam daily, combined ZOL and meloxicam, or vehicle. ZOL inhibited osteoclastic bone resorption at the tumour-bone interface. Meloxicam was more effective than ZOL at reducing xenograft growth but did not affect osteoclastic bone resorption. Although a synergistic effect of combined ZOL and meloxicam was not observed, combination therapy was well-tolerated and may be useful in the clinical management of bone-invasive feline OSCC.

Tumor microenvironment regulates metastasis and metastasis genes of mouse MMTV-PymT mammary cancer cells in vivo.

Metastasis is the primary cause of death in breast cancer patients, yet there are challenges to modeling this process in vivo. The goal of this study was to analyze the effects of injection site on tumor growth and metastasis and gene expression of breast cancer cells in vivo using the MMTV-PymT breast cancer model (Met-1 cells). Met-1 cells were injected into 5 sites (subcutaneous, mammary fat pad, tail vein, intracardiac, and intratibial), and tumors and metastases were monitored using bioluminescent imaging and confirmed with gross necropsy and histopathology. Met-1 tumors were analyzed based on morphology and changes in gene expression in each tissue microenvironment. There were 6 permissible sites of Met-1 tumor growth (mammary gland, subcutis, lung, adrenal gland, ovary, bone). Met-1 cells grew faster in the subcutis compared to mammary fat pad tumors (highest Ki-67 index). Morphologic differences were evident in each tumor microenvironment. Finally, 7 genes were differentially expressed in the Met-1 tumors in the 6 sites of growth or metastasis. This investigation demonstrates that breast cancer progression and metastasis are regulated by not only the tumor cells but also the experimental model and unique molecular signals from the tumor microenvironment.

Reprogramming of the tumour microenvironment by stromal PTEN-regulated miR-320.

PTEN (Phosphatase and tensin homolog deleted on chromosome 10) expression in stromal fibroblasts suppresses epithelial mammary tumours, but the underlying molecular mechanisms remain unknown. Using proteomic and expression profiling, we show that Pten loss from mammary stromal fibroblasts activates an oncogenic secretome that orchestrates the transcriptional reprogramming of other cell types in the microenvironment. Downregulation of miR-320 and upregulation of one of its direct targets, ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2) are critical events in Pten-deleted stromal fibroblasts responsible for inducing this oncogenic secretome, which in turn promotes tumour angiogenesis and tumour-cell invasion. Expression of the Pten-miR-320-Ets2-regulated secretome distinguished human normal breast stroma from tumour stroma and robustly correlated with recurrence in breast cancer patients. This work reveals miR-320 as a critical component of the Pten tumour-suppressor axis that acts in stromal fibroblasts to reprogramme the tumour microenvironment and curtail tumour progression.

Bone-invasive oral squamous cell carcinoma in cats: pathology and expression of parathyroid hormone-related protein.

Feline oral squamous cell carcinoma (OSCC) is the most common oral tumor in cats. There is no effective treatment, and the average duration of survival after diagnosis is only 2 months. Feline OSCC is frequently associated with osteolysis; however, the mechanisms responsible are unknown. The objective of this study was to characterize the epidemiology and pathology of bone-invasive OSCC in cats and to determine the expression of select bone resorption agonists. In sum, 451 cases of feline OSCC were evaluated. There was no sex or breed predisposition, although there were more intact cats in the OSCC group compared to the control group. Gingiva was the most common site, followed by the sublingual region and tongue. Cats with lingual OSCC were younger (mean, 11.9 years) compared to cats with gingival OSCC (mean, 13.6 years). In addition to osteolysis, there was periosteal new bone formation, osseous metaplasia of tumor stroma, and direct apposition of OSCC to fragments of bone, suggestive of bone-binding behavior. Eighty-two cases were selected for immunohistochemical detection of parathyroid hormone-related protein (PTHrP). Specimens with osteolysis had increased PTHrP expression and nuclear localization, compared to OSCC without osteolysis. Thirty-eight biopsies of OSCC with osteolysis were evaluated for tumor necrosis factor α expression, and only 4 biopsies had such expression in a small proportion of tumor cells. Increased tumor expression of PTHrP and increased localization of PTHrP to the nucleus were associated with osteolysis and may play an important role in bone resorption and tumor invasion in cats with OSCC.

Cloning, comparative sequence analysis and mRNA expression of calcium-transporting genes in horses.

Epithelial calcium transport occurs by paracellular and transcellular mechanisms. Transcellular transport in intestinal and renal epithelia involves several transport proteins, including transient receptor potential vanilloid member 5 (TRPV5), member 6 (TRPV6), calbindin D9k (CB9), calbindin D28k (CB28), sodium calcium exchanger 1 (NCX1), plasma membrane calcium ATPase 1 (PMCA1), and the vitamin D receptor (VDR). We are interested in the horse because of its unique calcium physiology (high blood calcium, high intestinal calcium absorption, high renal excretion of calcium, low vitamin D concentrations), and because horses often have dysregulated calcium balance with various diseases. We cloned the mRNA for equine TRPV5, TRPV6, CB9, CB28, NCX1, PMCA1, and VDR, performed comparative mRNA and protein sequence analysis, and quantified their mRNA expression in the kidney and gastrointestinal tract. Sequence homology for the mRNAs and proteins was high among mammals (>75%), with fish having the lowest homology (<75%). TRPV5, TRPV6, and CB9 expression was higher in the duodenum and proximal jejunum and followed a similar expression pattern. CB28 expression was greatest in the kidney. PMCA1 and NCX1 expression was similar throughout the intestine, but in the kidney PMCA1 expression was higher. Based on our findings, the proximal small intestine is the main site for transcellular calcium transport, with TRPV6 and CB9 serving as the main transport proteins. In the kidney, TRPV6, CB28, and PMCA1 are likely more important. The low VDR expression in the equine small intestine and kidney relative to the large intestine, together with the reported high intestinal absorption and renal excretion of calcium, and low vitamin D concentrations suggests that epithelial calcium transport in horses is not as dependent on vitamin D as in other species.

Rapid calcitonin response to experimental hypercalcemia in healthy horses.

Calcium has important physiological functions, and disorders of calcium homeostasis are frequent in horses. We have made important progress understanding equine calcium homeostasis; however, limited information on equine calcitonin (CT) is available, in part because of the lack of validated CT assays. To determine the CT response to high ionized calcium (Ca(2+)) concentrations in healthy horses, we induced hypercalcemia in 10 healthy horses using a calcium gluconate 23% solution (5mg/kg; 120 mL/500 kg horse) infused over 4 min. Four horses were infused with 120 mL of 0.9% NaCl and used as controls. We validated a human-specific CT radioimmunoassay for use in horses. Serum Ca(2+) concentrations increased from 6.2+/-0.3mg/dL to 9.9+/-0.5mg/dL (4 min; P<0.01). Serum CT increased from 16.7+/-8.0 pg/mL to 87.1+/-55.8 pg/mL at 2 min, and 102.5+/-51.1 pg/mL at 4 min (P<0.01). Serum CT returned to baseline by 20 min, whereas serum Ca(2+) returned to baseline by 40 min. Of interest, CT concentrations returned to baseline despite hypercalcemia, suggesting thyroid gland C-cell CT depletion. Resting CT values higher than 40 pg/mL were considered abnormally elevated. No significant changes in serum Ca(2+) or CT concentrations were found in control horses. The coefficients of variation for the CT radioimmunoassay were lower than 11.9%. We conclude that the equine thyroid gland C-cell responds quickly to changes in extracellular Ca(2+) concentrations by secreting large quantities of CT into the systemic circulation, indicating that CT is important in equine calcium homeostasis. The human CT radioimmunoassay can be used to measure changes in equine CT.

NOD/SCID mouse model of canine T-cell lymphoma with humoral hypercalcaemia of malignancy: cytokine gene expression profiling and in vivo bioluminescent imaging.

Lymphoma is a malignant neoplasm arising from B or T lymphocytes. In dogs, one-third of lymphomas are highly aggressive multicentric T-cell lymphomas that are often associated with humoral hypercalcaemia of malignancy (HHM). There are no cell lines or animal models to investigate the pathogenesis of T-cell lymphoma and HHM in dogs. We developed the first xenograft model by injecting lymphoma cells from an Irish Wolfhound intraperitoneally into NOD/SCID mice. The mice developed multicentric lymphoma along with HHM and increased parathyroid hormone-related protein (PTHrP) as occurs in dogs with T-cell lymphoma. Using cytokine complementary DNA arrays, we identified genes that have potential implications in the pathogenesis of T-cell lymphoma. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of T-cell lymphoma samples from hypercalcaemic canine patients showed that PTHrP likely plays a central role in the pathogenesis of HHM and that hypercalcaemia is the result of a combinatorial effect of different hypercalcaemic factors. Finally, we monitored in vivo tumour progression and metastases in the mouse model by transducing the lymphoma cells with a lentiviral vector that encodes a luciferase-yellow fluorescent protein reporter and showed that in vivo trafficking patterns in this model were similar to those seen in dogs. This unique mouse model will be useful for translational research in lymphoma and for investigating the pathogenesis of T-cell lymphoma and HHM in the dog.

Transcriptional regulation of parathyroid hormone-related protein promoter P2 by NF-kappaB in adult T-cell leukemia/lymphoma.

Parathyroid hormone-related protein (PTHrP) plays a primary role in the development of humoral hypercalcemia of malignancy (HHM) that occurs in the majority of patients with adult T-cell leukemia/lymphoma (ATLL) due to human T-cell lymphotropic virus type-1 (HTLV-1) infection. We previously showed that ATLL cells constitutively express high levels of PTHrP via activation of promoters P2 and P3, resulting in HHM. In this study, we characterized a nuclear factor-kappaB (NF-kappaB) binding site in the P2 promoter of human PTHrP. Using electrophoretic mobility shift assays, we detected a specific complex in Tax-expressing human T cells composed of p50/c-Rel, and two distinct complexes in ATLL cells consisting of p50/p50 homodimers and a second unidentified protein(s). Chromatin immunoprecipitation assays confirmed in vivo binding of p50 and c-Rel on the PTHrP P2 promoter. Using transient co-transfection with NF-kappaB expression plasmids and PTHrP P2 luciferase reporter-plasmid, we showed that NF-kappaB p50/p50 alone and p50/c-Rel or p50/Bcl-3 cooperatively upregulated the PTHrP P2 promoter. Furthermore, inhibition of NF-kappaB activity by Bay 11-7082 reduced PTHrP P2 promoter-initiated transcripts in HTLV-1-infected T cells. In summary, the data demonstrated that transcriptional regulation of PTHrP in ATLL cells can be controlled by NF-kappaB activation and also suggest a Tax-independent mechanism of activation of PTHrP in ATLL.

Inhibition of epidermal growth factor receptor signalling reduces hypercalcaemia induced by human lung squamous-cell carcinoma in athymic mice.

The purpose of this study was to evaluate the role of the epidermal growth factor receptor (EGFR) in parathyroid hormone-related protein (PTHrP) expression and humoral hypercalcaemia of malignancy (HHM), using two different human squamous-cell carcinoma (SCC) xenograft models. A randomised controlled study in which nude mice with RWGT2 and HARA xenografts received either placebo or gefitinib 200 mg kg(-1) for 3 days after developing HHM. Effectiveness of therapy was evaluated by measuring plasma calcium and PTHrP, urine cyclic AMP/creatinine ratios, and tumour volumes. The study end point was at 78 h. The lung SCC lines, RWGT2 and HARA, expressed high levels of PTHrP mRNA as well as abundant EGFR protein, but very little erbB2 or erbB3. Both lines expressed high transcript levels for the EGFR ligand, amphiregulin (AREG), as well as, substantially lower levels of transforming growth factor-alpha (TGF-alpha), and heparin binding-epidermal growth factor (HB-EGF) mRNA. Parathyroid hormone-related protein gene expression in both lines was reduced 40-80% after treatment with 1 muM of EGFR tyrosine kinase inhibitor PD153035 and precipitating antibodies to AREG. Gefitinib treatment of hypercalcaemic mice with RWGT2 and HARA xenografts resulted in a significant reduction of plasma total calcium concentrations by 78 h. Autocrine AREG stimulated the EGFR and increased PTHrP gene expression in the RWGT2 and HARA lung SCC lines. Inhibition of the EGFR pathway in two human SCC models of HHM by an anilinoquinazoline demonstrated that the EGFR tyrosine kinase is a potential target for antihypercalcaemic therapy.

3-Phosphoinositide-dependent protein kinase-1/Akt signalling and inhibition in a canine prostate carcinoma cell line.

Deregulation of the 3-phosphoinositide-dependent protein kinase-1 (PDK-1)/Akt signalling pathway is associated with prostate cancer development and progression. Inhibition of PDK-1/Akt signalling can be achieved using structurally optimized celecoxib derivatives such as OSU-03012. In this study, we treated the novel canine prostate cancer cell line, Ace-1, with OSU-03012 or dimethyl sulphoxide in vitro. We found that Akt was constitutively phosphorylated in the canine prostate cancer cell line Ace-1 and that there was a dose-dependent decrease in cell viability, and Akt and glycogen synthase kinase-3beta phosphorylation, in response to OSU-03012 treatment. This was accompanied by a dose-dependent increase in apoptosis. These data suggest that Akt signalling pathway inhibition is a potential strategy for the treatment of dogs with prostate cancer and that canine prostate cancer is a relevant large animal model for evaluating Akt pathway inhibitors such as OSU-03012 for use in people.

The effect of zoledronic acid and osteoprotegerin on growth of human lung cancer in the tibias of nude mice.

The pathogenesis of bone metastases may require the activation of osteoclasts by tumor-secreted factors, which promote important interactions with the bone microenvironment. We utilized an intratibial model of bone metastasis with bioluminescent imaging (BLI) to measure the effect of osteoclast inhibition on the interaction of human lung cancer cells with bone, and on tumor growth. Mice were injected with luciferase-transduced tumor cells (HARA, human pulmonary squamous carcinoma) and divided into three groups: (1) untreated, (2) twice weekly treatment with the bisphosphonate zoledronic acid (ZOL), or (3) osteoprotegerin (OPG). Histomorphometry and imaging were used to evaluate tumor burden, and parameters of osteoclast activity. Mice in the treated groups had increased bone density and decreased osteoclast numbers in nontumor-bearing tibiae. There was greater than 60% reduction in mean tumor volume in both treatment groups when evaluated by histomorphometry (P = 0.06 [OPG], P = 0.07 [ZOL]). However, bioluminescent imaging failed to show a reduction in tumor burden due to wide variability in the data. Osteoclast numbers along tumor-associated bone were significantly increased compared to tumor-free bone, and were not reduced by either treatment. Plasma calcium concentration was increased in all groups. Plasma tartrate-resistant acid phosphatase 5b was reduced in both treatment groups. Plasma PTHrP was significantly increased in the untreated tumor-bearing group, but was not significantly different in the two treatment groups compared to normal mice. OPG or ZOL did not change tumor cell proliferation, but ZOL increased HARA cell apoptosis. OPG and ZOL reduced tumor growth in the tibiae of treated mice, however, PTHrP production by HARA cells may have resulted in a high concentration in the bone microenvironment, partially overriding the antiosteoclast effects of both OPG and ZOL.

Feline head and neck squamous cell carcinoma: a natural model for the human disease and development of a mouse model.

Head and neck squamous cell carcinoma (H/N SCC) is a devastating disease in humans and cats, and shares similar features between the two species. The large population of pet cats in the United States, along with the high incidence of oral SCC in the cat, makes the cat an attractive candidate as a natural model for the human disease. There are similarities in pathology, progression, outcome, resistance to treatment, possible aetiologies and p53 expression, and we discuss the benefits of the cat as a natural model. We describe the development of a nude mouse xenograft model of feline oral SCC using the SCCF1 cell line transfected with a luciferase expression construct. In vivo tumour growth and metastasis were measured using serial bioluminescent imaging, and tumours grew best in the subcutis. The cat and nude mouse models will be useful to investigate the pathogenesis and the molecular basis of H/N SCC, and for preclinical drug screening.

Transcriptional regulation of parathyroid hormone-related protein promoter P3 by ETS-1 in adult T-cell leukemia/lymphoma.

Parathyroid hormone-related protein (PTHrP) plays a primary role in the development of humoral hypercalcemia of malignancy seen in the majority of adult T-cell leukemia/lymphoma (ATLL) patients with human T-cell lymphotropic virus type-1 (HTLV-1) infection. HTLV-1 Tax has been shown to complex with ETS-1 and SP1 to transactivate the PTHrP P3 promoter. Previously, we established a SCID/bg mouse model of human ATL with RV-ATL cells and showed that PTHrP expression was independent of Tax. In this study, we report an inverse correlation of PTHrP with tax/rex mRNA in multiple HTLV-1-positive cell lines and RV-ATL cells. Stimulation of Jurkat T cells with PMA/ionomycin upregulated the PTHrP P3 promoter by a previously characterized Ets binding site and also induced protein/DNA complex formation identical to that observed in RV-ATL cells. Further, we provide evidence that cotransfection with Ets-1 and constitutively active Mek-1 in HTLV-1-negative transformed T cells with stimulation by PMA/ionomycin not only resulted in a robust induction of PTHrP P3 but also formed a complex with ETS-1/P3 EBS similar to that in ATLL cells. Our data demonstrate that transcriptional regulation of PTHrP in ATLL cells can be controlled by T-cell receptor signaling and the ETS and MAPK ERK pathway in a Tax-independent manner.

Alternative splicing of parathyroid hormone-related protein mRNA: expression and stability.

Parathyroid hormone-related protein (PTHrP) is a multifunctional protein that is often dysregulated in cancer. The human PTHrP gene is alternatively spliced into three isoforms, each with a unique 3'-untranslated region (3'-UTR), encoding 139, 173 and 141 amino acid proteins. The regulation of PTHrP mRNA isoform expression has not been completely elucidated, but it may be affected by transforming growth factor-beta1 (TGF-beta1). In this study, we examined differences in the PTHrP mRNA isoform expression in two squamous carcinoma cell lines (SCC2/88 and HARA), an immortalized keratinocyte cell line (HaCaT), and spontaneous human lung cancer with adjacent normal tissue. In addition, the effect of TGF-beta1 on PTHrP mRNA isoform expression and stability was examined. Cell-type specific expression of PTHrP mRNA isoforms occurred between the various cell lines, normal human lung, and immortalized human keratinocytes (HaCaT). PTHrP isoform expression pattern was significantly altered between normal lung tissue and the adjacent lung cancer. In vitro studies revealed that TGF-beta1 differentially altered the mRNA steady-state levels and mRNA stability of the PTHrP isoforms. Protein-RNA binding studies identified different proteins binding to the 3'-UTR of the PTHrP isoforms (139) and (141), which may be important in the differential mRNA stability and response to cytokines between the PTHrP isoforms. The data demonstrate that there is cell-type specific expression of PTHrP mRNA isoforms, and disruption of the normal regulation during cancer progression may in part be associated with TGF-beta1-induced changes in PTHrP mRNA isoform expression and stability.

Effects of 1,25(OH)2D3, 25OHD3, and EB1089 on cell growth and Vitamin D receptor mRNA and 1alpha-hydroxylase mRNA expression in primary cultures of the canine prostate.

The aim of this study was to investigate effects of 1,25(OH)(2)D(3) (calcitriol), 25OHD(3), and EB1089 on cell growth and on Vitamin D receptor (VDR) mRNA and 1alpha-hydroxylase (1alpha-OHase) mRNA expression in normal canine prostatic primary cultures. Canine prostatic epithelial cells were isolated, cultured, and treated with vehicle (ethanol), calcitriol, 25OHD(3), and EB1089 at 10(-9) and 10(-7)M. The VDR was present in epithelial and stromal cells of the canine prostate gland. 1,25(OH)(2)D(3), 25OHD(3), and EB1089 inhibited epithelial cell growth at 10(-7)M compared to vehicle-treated controls [calcitriol (P < 0.01), EB1089 (P < 0.01), and 25OHD(3) (P < 0.05)]. Epithelial cells treated with calcitriol and EB1089 at 10(-7)M had slightly increased VDR mRNA expression (0.2-0.3-fold) at 6 and 12h compared to controls. There was no difference in 1alpha-OHase mRNA expression in epithelial cells treated with these three compounds. 1,25(OH)(2)D(3) and its analogs may be effective antiproliferative agents of epithelial cells in certain types of prostate cancer.

Benign prostate hyperplasia: evaluation of treatment response with DCE MRI.

Benign prostate hyperplasia (BPH) is a major disease and its non-surgical therapy a major area of interest. The purpose of this study was to establish perfusion parameters in beagles with BPH using dynamic contrast-enhanced (DCE) MRI and to investigate changes due to the effects of finasteride treatment. Twelve male beagles (mean age 4.4 +/- 0.9,years) were divided into a control and treatment group that received a daily dose of 1 mg/kg finasteride. DCE MRI was carried out in a clinical scanner using a 3D spoiled gradient echo sequence prior to and during treatment. 0.2 mmol/kg contrast agent (gadoteridol) was administered with an injection rate of 0.2 ml/s followed by a 15 ml flush of saline. Contrast enhancement was evaluated by pharmacokinetic mapping of a two-compartment model with colour overlay images in addition to regional ROI analysis. Quantitative parameters were defined by the amplitude of contrast enhancement A, the exchange rate k(ep) and the time to maximum signal enhancement. Dynamic contrast-enhanced MRI investigations of the prostate revealed two distinct zones, an inner, periurethral zone and an outer, parenchymal zone. The periurethral zone is highly vascularized, whereas the parenchymal zone is moderately vascularized when compared to other parenchymal organs. During treatment, in the parenchymal zone the intensity of enhancement (amplitude A) and the time to maximum signal enhancement increased, while the exchange rate k(ep) decreased. Dynamic contrast-enhanced MRI of BPH reveals distinct differences between individual zones within the prostate. Moreover, changes during successful treatment suggest increased blood volume per volume of tissue and decreased vessel leakiness.