Phase II study of interim PET-CT-guided response-adapted therapy in advanced Hodgkin's lymphoma.

BACKGROUND: Combination chemotherapy ABVD (doxorubicin, bleomycin, vinblastine and dacarabazine) cures ∼70% of patients with advanced Hodgkin's lymphoma (aHL, stages IIB, III and IV) while more toxic escalated BEACOPP (EB, combination of bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisolone) increases cure rates to 85%. Patients with a positive interim positron emission tomography-computerized tomography (PET-CT) scan after two cycles (PET-2) of ABVD have very poor outcomes with continued ABVD. Intensifying therapy with EB in PET-2-positive patients ('response-adapted therapy') may improve cure rates, whereas the negative patients can continue ABVD alone. PATIENTS AND METHODS: Eligible patients with newly diagnosed aHL received two cycles of ABVD and underwent PET-2 (scored with semi-quantitative 5-point visual criteria, 'Deauville score'). PET-2-negative patients continued four additional cycles of ABVD, whereas PET-2-positive patients received four cycles of EB. A phase II sample size of 50 was estimated keeping the lower and higher proportion of rejection of the event-free survival (EFS) as 70% and 85%, respectively. RESULTS: Fifty patients [median age 28 (12-60) years; male : female: 39 : 11; stages: IIB-3 (6%), III-29 (58%) and IV-18 (36%); International Prognostic Score (IPS): 0-3: 34 (68%); 4-7: 16 (32%)] were enrolled; 49 underwent PET-2. Eight (16%) were PET-2-positive, whereas 41 (84%) were negative. Forty-seven were evaluable for EFS and all 50 for overall survival (OS). The 2-year EFS was 76% (95% CI: 68-83) and OS was 88% (95% CI: 82-94). PET-2 was strongly prognostic-2-year EFS, negative versus positive: 82% versus 50%; P = 0.013. CONCLUSION: PET-2 response-adapted strategy could not achieve EFS of 85% in aHL. However, escalated therapy improved outcomes in PET-2-positive patients compared with historical data. TRIAL REGISTRATION: CTRI/2012/06/002741 (http://www.ctri.nic.in) and NCT01304849 (http://www.clinicaltrials.gov).

Multiscale topology optimization of pelvic bone for combined walking and running gait cycles.

We propose a multiscale topology optimization procedure of pelvic bone using weighted compliance minimization. In macroscale optimization, a level set-based method is used, which gives a binary structure. In microscale optimization, cubic lattice-based homogenization is done while keeping the global geometry fixed. For the macroscale, a volume constraint equal to the volume of the pelvic bone is imposed, whereas, for the microscale, a mass constraint equal to the mass of the pelvic bone is imposed. The optimal geometries are compared with pelvic bone using different metrics and show good similarity with the same. Designed geometries are additively manufactured and experimentally tested for stiffness.

Assessment of an ensemble of machine learning models toward abnormality detection in chest radiographs.

Respiratory diseases account for a significant proportion of deaths and disabilities across the world. Chest X-ray (CXR) analysis remains a common diagnostic imaging modality for confirming intra-thoracic cardiopulmonary abnormalities. However, there remains an acute shortage of expert radiologists, particularly in under-resourced settings, resulting in severe interpretation delays. These issues can be mitigated by a computer-aided diagnostic (CADx) system to supplement decision-making and improve throughput while preserving and possibly improving the standard-of-care. Systems reported in the literature or popular media use handcrafted features and/or data-driven algorithms like deep learning (DL) to learn underlying data distributions. The remarkable success of convolutional neural networks (CNN) toward image recognition tasks has made them a promising choice for automated medical image analyses. However, CNNs suffer from high variance and may overfit due to their sensitivity to training data fluctuations. Ensemble learning helps to reduce this variance by combining predictions of multiple learning algorithms to construct complex, non-linear functions and improve robustness and generalization. This study aims to construct and assess the performance of an ensemble of machine learning (ML) models applied to the challenge of classifying normal and abnormal CXRs and significantly reducing the diagnostic load of radiologists and primary-care physicians.

Management outcomes of pediatric and adolescent papillary thyroid cancers with a brief review of literature.

BACKGROUND: Papillary carcinoma of thyroid (PTC) is a rare disease in children and adolescents and contributes to about 1.5%-3% of all pediatric malignancies. To date, no randomized trial has ever been performed in the pediatric population and management of these patients has been extrapolated from adult practice. MATERIALS AND METHODS: Retrospective analysis of the patients treated for PTC in the age <21 years, between the years 1998-2013 at a tertiary cancer center from India. RESULTS: Sixty-seven patients were treated in the above said period with a male:female ratio of 1:1.6 and a median age of 18 years. Fifty-two (77.6%) patients clinically presented as a thyroid swelling with or without nodal swelling while 13 (19.4%) presented with isolated nodal swelling. Surgery was performed in 30 patients at a nononcological hospital and was subsequently referred to our center; more than half of them needed a completion surgery at our center. Pathologically, multifocal tumors were found in close to a quarter of the patients. Among the pathological variants, classical, follicular, and tall cell variants comprised 65.7%, 28.4%, and 5.9% of the cases, respectively. Nodal positivity was noted 71.6% of the cases of which 14.5% were N1a disease and the vast majority (85.5%) harboring N1b disease. The median follow-up period of the study cohort was 104 months during which there were 3 local, 6 nodal, and 2 systemic recurrences. The 5- and 10-year disease-free survival were found to be 85.9% and 81.4%, respectively. Univariate and multivariate analysis has shown no significant clinical and pathological feature defining the disease outcomes except for the T-stage. Logistic regression revealed extrathyroidal invasion and the age ≤ 15 years correlated with nodal positivity. CONCLUSION: Being a rare malignancy, pediatric and adolescent PTCs tend to behave differently from adult PTC with a seemingly aggressive clinical presentation; however, they are associated with excellent survival outcomes.

A novel stacked generalization of models for improved TB detection in chest radiographs.

Chest x-ray (CXR) analysis is a common part of the protocol for confirming active pulmonary Tuberculosis (TB). However, many TB endemic regions are severely resource constrained in radiological services impairing timely detection and treatment. Computer-aided diagnosis (CADx) tools can supplement decision-making while simultaneously addressing the gap in expert radiological interpretation during mobile field screening. These tools use hand-engineered and/or convolutional neural networks (CNN) computed image features. CNN, a class of deep learning (DL) models, has gained research prominence in visual recognition. It has been shown that Ensemble learning has an inherent advantage of constructing non-linear decision making functions and improve visual recognition. We create a stacking of classifiers with hand-engineered and CNN features toward improving TB detection in CXRs. The results obtained are highly promising and superior to the state-of-the-art.

Protective action of luminal bile salts in necrotizing acute pancreatitis in mice.

Bile salts in the intestinal lumen act to inhibit the release of cholecystokinin (CCK). Recent studies have shown that CCK may play a permissive role in the development of acute pancreatitis. In this study, the amount of luminal bile salts in female Swiss Webster mice was either decreased by feeding 4% (wt/wt) cholestyramine or increased by feeding 0.5% sodium taurocholate for 1 wk. Plasma levels of CCK were stimulated by cholestyramine and inhibited by taurocholate. Then, acute pancreatitis was induced either by caerulein injections, or by feeding a choline-deficient, ethionine-supplemented (CDE) diet. Feeding of cholestyramine significantly decreased survival from 25% to 0% in the CDE pancreatitis, and increased the magnitude of elevation of serum amylase levels and the extent of pancreatic necrosis in both models of pancreatitis; CCK-receptor blockade with CR-1409 completely abolished the adverse effects of cholestyramine. In contrast, feeding of taurocholate significantly increased survival to 100% and decreased the elevation of serum amylase and pancreatic necrosis; CCK-8 antagonized these actions of taurocholate. Luminal bile salts appear to provide a physiologic protection against necrotizing pancreatitis, at least in part, both by inhibiting the release of CCK and by promoting resistance of the pancreas to CCK excessive stimulation in vivo.

Long-term outcome of diffuse large B-cell lymphoma: Impact of biosimilar rituximab and radiation.

INTRODUCTION: Rituximab (R)-CHOP improves survival over CHOP in diffuse large B-cell lymphoma (DLBCL). The availability of biosimilar rituximab in India has increased access of this drug. We report on the impact of treatment on outcomes with special emphasis on the impact of biosimilar rituximab and radiation. METHODS: Outcomes of adults (age 15-60 years) treated with CHOP+/- Rituximab radiation were analyzed retrospectively to look at baseline features, treatment, and event-free and overall survival (EFS and OS). RESULTS: In the period 2000-2013, 444 patients (median age 47 years: 15-60; males: 288 [65%]; Stage III/IV: 224 [50%]; age-adjusted international prognostic index [aaIPI] Score 2 or 3 in 50%) received either CHOP (n = 325 [73%]) or RCHOP (n = 119 [27%]) therapy. Biosimilar rituximab and the original were used in 95 (80%) and 24 (20%) patients, respectively. Radiation was given in 134 (30%) patients (Stages I and II, 100/220 [45%] and Stages III and IV, 34/224 [15%]). After a median follow-up of 46 (0.2-126) months, the 5-year EFS and OS were 59% and 68%, respectively. The factors predicting inferior EFS and OS were age> 40 years, performance status 2-4, Stage III/IV, hemoglobin <12 g/dL, the aaIPI Score 2 or 3, and nonuse of rituximab and radiation. Radiation used in early stage disease benefitted all subgroups regardless of bulky disease, use of rituximab, or the number of cycles of chemotherapy. Addition of rituximab improved survival across all categories of aaIPI. CONCLUSION: Availability of biosimilar rituximab has increased access and survival of patients with DLBCL in India. Radiotherapy improved outcomes in early stages.

Primary transfection as a mechanism for transformation of host cells by human tumor cells implanted in nude mice.

Establishment of cell lines in vitro from a human lung cancer xenograft in nude mice resulted in transformed mouse cell lines. The transformed mouse cell lines expressed both mouse-specific and human-specific histocompatibility antigens. Of 3 cell lines, 2 were tumorigenic in BALB/c nude mice but not in normal mice. Tumors formed by the transformed mouse cell lines were fibroblastoid and epithelioid by histology. In addition, tumors exhibited neuroepithelial differentiation by ultrastructural and immunohistochemical analysis. Phenotypically they were similar to the original patient and human xenograft tumor. These data suggest that previous reports of host cell transformation and induction of fibrosarcomas may not be true fibrosarcomas. Human DNA sequences were present in the tumorigenic cell lines, indicating that spontaneous transfection of human tumor DNA into host cells had occurred. The implication of these findings is that human genetic information has been transferred to primary mouse host fibroblasts, which resulted in a transformed as well as a differentiated phenotype.

Gastrin gene expression is required for the proliferation and tumorigenicity of human colon cancer cells.

The majority of human colon cancers express the gastrin gene, and a significant percentage bind gastrin-like peptides. However, it is not known if gastrin gene products are physiologically relevant to the growth and proliferation of human colon cancers. To investigate the functional role of gastrin gene expression, we examined the effect of gastrin antisense (AS) RNA expression on the growth and tumorigenicity of colon cancer cells. The full-length human gastrin cDNA was cloned in the AS direction in a retroviral vector under the transcriptional control of human cytomegalovirus promoter. Three representative human colon cancer cell lines that expressed negligible (Colo-205A) to significant (Colo-320 and HCT-116) levels of gastrin mRNA were transfected with either AS or control vectors and subjected to various growth studies in vitro and in vivo. The proliferative and tumorigenic potential of the AS clones from the gastrin-expressing cell lines was significantly suppressed compared to that of the control clones, whereas the growth of Colo-205A-AS cells (the negative control) was similar to that of the Colo-205A-C-cells, indicating the relative specificity of the antitumorigenic effects of AS gastrin RNA expression. We believe that this is the first evidence that supports a possible critical role of gastrin gene expression in the tumorigenicity of human colon cancers that express the gastrin gene. Because > 60-80% of human colon cancers express the gastrin gene, it can be expected that the growth of a significant percentage of these cancers may be critically dependent on the expression of gastrin gene products. Therapeutic measures, such as the AS strategy used in the present study, may therefore prove to be useful in treating human colon cancers in the future.

Inhibition of human gastric adenocarcinoma xenograft growth in nude mice by alpha-difluoromethylornithine.

We studied the effect of inhibition of polyamine biosynthesis by alpha-difluoromethylornithine on the growth of a human gastric adenocarcinoma (CLEES) xenotransplanted in nude mice. CLEES is a well-differentiated gastric adenocarcinoma of the intestinal type. The doubling time has ranged from 7 to 10 days through 11 passages. Electron microscopic and immunohistochemical studies comparing the original tumor and xenotransplants showed similar structure and similar amounts of carcinoembryonic antigen. Polyamine biosynthesis is required for cell division. alpha-Difluoromethylornithine inhibits ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis. In this study, 48 athymic mice were used in two experiments. In the first experiment, two groups of 12 mice each were inoculated with CLEES tumor cells and received either tap water or a 3% alpha-difluoromethylornithine solution as drinking water. Tumor size was measured twice weekly. Tumor size was significantly decreased from controls by the fourth week of treatment and at all points of analysis thereafter for 7 wk. In the second experiment, alpha-difluoromethylornithine significantly reduced tumor concentrations of the polyamines putrescine and spermidine. In addition, the tumor content of DNA was significantly reduced in treated mice (0.64 +/- 0.16 mg) compared to controls (4.76 +/- 0.92 mg). Our data suggest that inhibition of polyamine biosynthesis may be a useful component of multidrug chemotherapy for human gastric adenocarcinoma. Establishment of tumor lines such as this gastric adenocarcinoma will facilitate further studies on the biological behavior of human gastric cancer and its response to chemotherapeutic manipulation in vivo.

Isolation and characterization of a 30 kDa membrane glycoprotein from human stratum corneum.

Using iodinated concanavalin A in conjunction with gel electrophoresis, we have identified a 30 kDa glycoprotein in the stratum corneum of human skin. We isolated this glycoprotein by extraction in nonionic detergent, affinity chromatography and preparative gel electrophoresis. It binds to concanavalin A but not to three other lectins. The purified glycoprotein migrates at 30 kDa whether or not reducing agents are present. It is rich in histidine and lysine, but lacks arginine, proline, tyrosine and methionine. It is clearly distinct from filaggrin. We prepared a monospecific polyclonal antibody to this glycoprotein and localized it by immunohistochemistry exclusively to the cell membrane of corneocytes. We postulate that the glycoprotein may play a role in the cohesion and desquamation of corneocytes.

An allelic series of mutations in the kit ligand gene of mice. I. Identification of point mutations in seven ethylnitrosourea-induced Kitl(Steel) alleles.

An allelic series of mutations is an extremely valuable genetic resource for understanding gene function. Here we describe eight mutant alleles at the Steel (Sl) locus of mice that were induced with N-ethyl-N-nitrosourea (ENU). The product of the Sl locus is Kit ligand (or Kitl; also known as mast cell growth factor, stem cell factor, and Steel factor), which is a member of the helical cytokine superfamily and is the ligand for the Kit receptor tyrosine kinase. Seven of the eight ENU-induced Kitl(Sl) alleles, of which five cause missense mutations, one causes a nonsense mutation and exon skipping, and one affects a splice site, were found to contain point mutations in Kitl. Interestingly, each of the five missense mutations affects residues that are within, or very near, conserved alpha-helical domains of Kitl. These ENU-induced mutants should provide important information on structural requirements for function of Kitl and other helical cytokines.

An allelic series of mutations in the Kit ligand gene of mice. II. Effects of ethylnitrosourea-induced Kitl point mutations on survival and peripheral blood cells of Kitl(Steel) mice.

The ligand for the Kit receptor tyrosine kinase is Kit ligand (Kitl; also known as mast cell growth factor, stem cell factor, and Steel factor), which is encoded at the Steel (Sl) locus of mice. Previous studies revealed that Kitl(Sl) mutations have semidominant effects; mild pigmentation defects and macrocytic, hypoplastic anemia occur in heterozygous mice, and more severe pigmentation defects and anemia occur in homozygotes. Lethality also occurs in mice homozygous for severe Kitl(Sl) mutations. We describe the effects of seven new N-ethyl-N-nitrosourea (ENU)-induced Kitl(Sl) mutations and two previously characterized severe Kitl(Sl) mutations on pigmentation, peripheral blood cells, and mouse survival. Mice heterozygous for each of the nine mutations had reduced coat pigmentation and macrocytosis of peripheral blood. In the case of some of these mutations, however, red blood cell (RBC) counts, hemoglobin concentrations, and hematocrits were normal in heterozygotes, even though homozygotes exhibited severely reduced RBC counts and lethality. In homozygous mice, the extent of anemia generally correlates with effects on viability for most Kitl(Sl) mutations; i.e., most mutations that cause lethality also cause a more severe anemia than that of mutations that allow viability. Interestingly, lethality and anemia were not directly correlated in the case of one Kitl(Sl) mutation.

Effect of oxygen on the clonal growth of adherent cells (CFU-F) from different compartments of mouse bone marrow.

We have used collagenase to isolate cell populations from different compartments of mouse bone marrow. Cells were obtained from the shaft of the femur, the endosteum, and compact bone. We have studied the growth-enhancing effects of physiologically low oxygen levels on fibroblast colony-forming unit (CFU-F) growth in vitro. Low oxygen levels (0.1%-10% O2) increased CFU-F formation 1.8- to 2.8-fold. However, cells from the compact bone consistently grew with much higher efficiencies (12- to 31-fold) than did cells from femoral cavity and endosteal areas. These data indicate the usefulness of enzymatic methods of isolating cells from compartments of bone and the use of low oxygen atmosphere to enhance stromal cell growth in vitro.

Characterization of heterotypic adherence between transformed human lymphoblastic cells and marrow stromal cells: VCAM-1 is a ligand for one of the leukemia cell adhesion proteins.

Marrow stromal cells are important in normal myelopoiesis and support growth of leukemia/lymphoma (LL) cells in vitro. We have previously described the heterotypic adherence of a human B-lymphoblastic cell line (UTMB-460) to marrow stromal cells (MSC). We have extended these observations to a human T-lymphoblastic cell line (CEM) and characterized the heterotypic adherence of B- and T-lymphoblastic cell lines to human MSC. Electron microscopy demonstrated UTMB-460 cells were in very close apposition to the MSC, but no specific intercellular junctions were noted. Under the conditions employed, these MSC express extracellular fibronectin, collagen types I and IV, intracellular laminin, and vimentin, but no factor VIII-R antigen. In addition, the MSC had receptors for the lectin Ulex europaeus agglutinin I. UTMB-460 and CEM cells do not adhere to extracellular matrix (ECM) proteins secreted by the MSC, i.e., fibronectin, collagen types I, III, or IV, or laminin. Monoclonal antibodies (MoAbs) against CD11a, CD11b, CD18, and CD54 and a polyclonal anti-human fibronectin antibody do not inhibit attachment of either B- or T-lymphoblastic cells to MSC. Peptides GRGES and GRGDS did not inhibit adherence of UTMB-460 and CEM cells to MSC. In contrast, the anti-vascular cell adhesion molecule (VCAM)-1 MoAb (4b9) caused significant inhibition (p < 0.01) of the adherence of both UTMB-460 and CEM cells to normal human MSC monolayers. These data suggest: (1) that MSC to which lymphoblastic cells adhere are specialized mesenchymal cells; (2) that the membrane interactions between T- and B-lymphoblastic cells and MSC involve close apposition of cell membranes of MSC and the lymphoblastic cells; (3) that the heterotypic adherence between B- and T-lymphoblastic cell lines (UTMB-460 and CEM) and MSC does not involve the RGD recognition sequence of the integrin family, the B2 leukocyte integrins, CD44, LAM-1, or the ECM proteins examined; and (4) that VCAM-1 may at least be partially responsible for heterotypic adherence between human MSC and B- and T-lymphoblastic cells.

Heterotypic adherence between murine leukemia/lymphoma cells and marrow stromal cells involves a recognition mechanism with galactosyl and mannosyl specificities.

We have previously shown that leukemia/lymphoma (LL) cells adhere to marrow stromal cells (MSC), and MSC induce clonal growth of LL cells. Even though CD11a/18 and CD54 are important in leukocyte and endothelial cell interaction, the literature suggested that these adhesion proteins are not involved in adhesion of hematopoietic stem cells to MSC. We therefore utilized a unique ICAM-1- murine MSC line (MLT) to evaluate the mechanisms of adherence of LL cells (L5178Y and L1210) to MLT. Adherence of LL cells to extracellular matrix (ECM) proteins was also examined. L1210 cells attached to collagen types III and IV, laminin, and fibronectin, but not to collagen type I. L5178Y cells did not attach to any of the ECM proteins tested. The adherence of both L1210 and L5178Y to MSC was unaffected by rat monoclonal antibodies to murine CD11a, CD11b, and CD18. Neoglycoprotein probes, mannosyl-bovine serum albumin (BSA) and galactosyl-BSA, inhibited the adherence of L5178Y and L1210 cells to MSC by 34%-63% of controls at concentrations of 10(-3) and 5 x 10(-3) M. In contrast, fucosyl-BSA had no inhibitory effect on LL cell adherence of MLT. These data suggest that 1) LL cells may adhere to MSC by a lectin mechanism with mannosyl and galactosyl specificities; and 2) other mechanisms of adherence, not yet defined, are also important in this system.

Culture of basal cell carcinoma.

Studies of basal cell carcinoma have been hindered by a lack of a suitable and reproducible tissue-culture model system. We have succeeded in growing this tumor in primary culture from eight different patients. We can separate and grow the tumor cells and the stromal cell component. We also culture normal keratinocytes and fibroblasts from the same patient for comparative studies. All the cell types have been subcultured four to five times and cryopreserved. The normal keratinocytes were indistinguishable from the tumor cells in ploidy, in rate of growth, and in the failure to express ICAM-1. Both cell types also fail to synthesize the matrix proteins: types I and IV collagens. Differences were noted in the expression of fibronectin and the bullous pemphigoid antigen, with the normal cells expressing the antigens although the tumor cells did not. Interferons exogenously added to the culture media preferentially killed the basal cell carcinoma cells, as compared to normal keratinocytes from the same patients. We believe that our culture system opens possibilities for biochemical and molecular studies of this disease, and for in vitro testing of antitumor agents for clinical therapy.

Recombinant desmoglein 3 has the necessary epitopes to adsorb and induce blister-causing antibodies.

The development of an animal model for studying the pathogenesis of pemphigus vulgaris (PV) has been hampered by the unavailability of the purified full-length autoantigen desmoglein 3 (Dsg 3).Therefore, we expressed Dsg 3 using a baculovirus expressed system. The expressed protein was identified as Dgs 3 by its reactivity with a pan-cadherin anti-serum, an anti-serum to a Dsg 3 synthetic peptide, or patient serum, and by amino-terminal sequencing. Carbohydrate analysis showed that recombinant Dsg 3 was glycosylated. While a majority of the recombinant protein was cell associated, by immunoprecipitation, some Dsg 3 was demonstrated in the medium. The Dgs 3 could adsorb out blister-causing antibodies from patient sera. Rabbit anti- Dsg 3 antibodies induced by the recombinant Dsg 3 showed specific binding to intercellular spaces of monkeys esophagus by indirect immunofluorescence. Moreover, these antibodies induced PV-like blisters in neonatal mice and weakly bound perilesional epidermis. Availability of large quantities of relatively pure Dsg 3 should now facilitate studies aimed at understanding Dsg 3 structure and pathogenesis of PV, with implications for developing specific immunotherapies.

Rat GST 8-8 is expressed predominantly in myeloid origin cells infiltrating the gravid uterus.

Previous studies from our laboratory have shown a relatively high expression of rGST8-8 in uterine tissues. This GST isozyme displays relatively high glutathione-peroxidase activity towards lipid-hydroperoxides and towards toxic 4-hydroxyalkenals generated from lipid peroxidation. Since the uterus is a unique organ, subject to oxidative stress due to infiltration by immune effector cells during gestation and because this infiltration is readily identifiable histologically, the studies reported herein were performed to localize the cell specific expression of rGST8-8 to determine whether immune effector cells infiltrating the pregnant rat uterus specifically expressed rGST8-8. A 75 bp end-radiolabeled cRNA probe was prepared from the full length mGSTA4-4 cDNA from the region which is highly homologous with rGST8-8. This cRNA probe was used for in situ hybridization studies to localize rGST8-8 in specific cell types of gravid rat uterus. Results of these studies indicate that this GST isozyme is selectively expressed in myeloid origin cells such as monocytes/macrophages, and neutrophils infiltrating the uterine endometrium and in vascular walls. Selective expression of rGST8-8 in the myeloid origin cells, which are known to generate higher levels of reactive oxygen species, suggests that this GST isozyme plays an important role in the protection mechanisms against lipid peroxidation.

Effects of parathyroid hormone-related peptide on adenosine 3',5'-monophosphate and ornithine decarboxylase in a human colonic cell line.

PTH-related peptide (PTHrP) is widely distributed in normal tissues, including the gut, and is considered a potential autocrine or paracrine regulator of cellular growth and differentiation. With this in mind, a human colonic cell line (LoVo) was used to study the effect of PTHrP on ornithine decarboxylase (ODC), because ODC is known to have profound effects on the growth and differentiation of many cell types via stimulation of synthesis of polyamines. cAMP also was measured, because this second messenger has been implicated in the regulation of ODC activity. Nearly confluent LoVo cells, grown in F-12 medium and 10% fetal bovine serum (FBS), were preincubated in 1% FBS for at least 5 h, and then PTHrP-(1-34) was added, and the incubation was continued for up to 6 h. Cell extracts were analyzed for ODC activity by measuring 14CO2 liberated from 14C-labeled ornithine, for cAMP by RIA, and for ODC mRNA by Northern analysis. PTHrP produced dose-related increases in both cAMP (2- to 3-fold) and ODC (3- to 5-fold), with a maximal effect at 0.1-1 microM and an ED50 of 1-10 nM. Comparison of the cAMP and ODC responses to PTHrP showed a strong correlation (r = 0.96; P less than 0.001). The effects of 1 microM PTHrP-(1-34) to increase cAMP and ODC were completely inhibited by 10-20 microM of the specific antagonist [Asn10,Leu11]PTHrP-(7-34). PTHrP-(1-34) did not stimulate ODC activity when cells were incubated without FBS. The stimulation of ODC activity by PTHrP-(1-34) was maximal at 2 h, a time at which an increase in ODC mRNA also was evident. PTH-(1-34) and forskolin also stimulated ODC activity, but PTHrP-(67-86) amide was ineffective. The results indicate that the N-terminal portion of the PTHrP molecule can stimulate ODC activity in a human colon cell line and that the effect is probably mediated by cAMP. The results are consistent with the idea that PTHrP may influence cell growth and differentiation in the gut via an effect on polyamine biosynthesis. Since LoVo cells also express PTHrP mRNA, this gastrointestinal cell line may serve as a useful model for studying autocrine regulation of gut cell growth and differentiation by PTHrP.