Visualizing genomic data: The mixing perspective.

We report on a novel way to visualize genomic data. By considering genome coding sequences, cds, as sets of the N=61 non-stop codons, one obtains a partition of the total number of codons in each cds. Partitions exhibit a statistical property known as mixing character which characterizes how mixed the partition is. Mixing characters have been shown mathematically to exhibit a partial order known as majorization (Ruch, 1975). In previous work (Seitz and Kirwan, 2022) we developed an approach that combined mixing and entropy that is visualized as a scatter plot. If we consider all 1,121,505 partitions of 61 codons, this produces a plot we call the theoretical mixing space, TGMS. A normalization procedure is developed here and applied to real genomic data to produce the genome mixing signature, GMS. Example GMS's of 19 species, including Homo sapiens, are shown and discussed.

Parathyroid hormone-related protein overexpression in the human colon cancer cell line HT-29 enhances adhesion of the cells to collagen type I.

The present experiments examined the potential ability of parathyroid hormone-related protein (PTHrP) to influence growth of the human colon cancer cell HT-29 and the ability of the cell to adhere to several extracellular matrix (ECM) proteins found in normal tissues. Addition of PTHrP analogs, PTHrP (1-34), PTHrP (67-86), or PTHrP (107-139), to HT-29 cells in culture did not influence cell growth or the adhesion of the cells to wells coated with fibronectin, laminin, or collagen type I. Likewise, in HT-29 cells induced to overexpress PTHrP by stable transfection with PTHrP cDNA, compared to vector-transfected control HT-29 cells, no effect on cell growth occurred. However, in the transfected cells, the increased production of PTHrP significantly enhanced cell adhesion to type I collagen but not to fibronectin or laminin. The results raise the possibility that PTHrP might play a role in colon tumor invasion and metastasis by influencing cell adhesion to specific extracellular matrix proteins.

Overexpression of parathyroid hormone-related protein promotes cell growth in the rat intestinal cell line IEC-6.

The rat intestinal cell line, IEC-6, was used as a model to study effects of parathyroid hormone-related protein (PTHrP) on crypt cell growth. Studies showed that addition of PTHrP analogs (1-34), (67-86), or (107-139) to growth medium did not affect proliferation of cells grown in either high (10% Nu-Serum) or low serum (1% Nu-Serum). However, studies on clonal lines of IEC-6 cells stably transfected with PTHrP cDNA and overexpressing PTHrP showed that increased PTHrP production enhanced cell growth and 3H-thymidine incorporation in high, but not low, serum. Additional studies examined the role of the nuclear localization sequence (NLS) of PTHrP in mediating the growth effect. In three clonal IEC-6 lines transfected with PTHrP cDNA bearing a mutated NLS, the ability of PTHrP to stimulate 3H-thymidine incorporation and cell growth was lost. The results suggest that endogenously produced PTHrP can promote proliferation of IEC-6 cells and that the integrity of the NLS of PTHrP is required for its growth effects.

Overexpression of parathyroid hormone-related protein enhances apoptosis in the rat intestinal cell line, IEC-6.

We used the rat intestinal cell line, IEC-6, to study potential effects of overexpression of PTH-related protein (PTHrP) on apoptosis. A clonal line of PTHrP-overexpressing cells was established by stably transfecting parental cells with PTHrP complementary DNA in a sense orientation (sense). A similarly transfected line stably, transfected with empty vector, served as control (vector). Immunoreactive PTHrP, measured in culture medium, showed that sense cells secreted approximately 30 times as much PTHrP as did vector control cells. Apoptosis induced by serum withdrawal was evaluated by several methods. DNA laddering was demonstrable in sense-transfected cells as early as 12 h after serum withdrawal but not until later time points in vector-transfected control cells. Flow cytometric analysis of propidium iodide-stained cells showed a greater increase in the sub-G1 (apoptotic) population in sense cells, compared with vector. Fluorescent microscopy with Hoechst 33258 dye showed increased nuclear fragmentation and condensation in sense cells. Studies of apoptotic gene expression by ribonuclease protection assay, and protein by Western blot analysis, showed an enhanced ratio of Bax to Bcl-x(L) in sense cells. Mutation of the PTHrP nuclear localization amino acid sequence negated the ability of PTHrP to enhance apoptosis.

Regulation of PTH/PTH-related protein receptor expression by endogenous PTH-related protein in the rat osteosarcoma cell line ROS 17/2.8.

We have utilized clonal lines of the rat osteoblastic cell line ROS 17/2.8 stably transfected with full-length parathyroid hormone-related protein (PTHrP) cDNA in a sense or an antisense orientation to examine the effects of alteration in the production of endogenous PTHrP on expression of the PTH/PTHrP receptor. In the stably transfected clonal cell lines, changes in PTH/PTHrP receptor expression were evaluated by Northern blot analysis, whole-cell ligand binding of 125I-[Tyr36] PTHrP (1-36), and exogenous PTHrP (1-34)-stimulated cyclic adenosine monophosphate (cAMP) accumulation. Compared to control (vector-transfected) cells, PTHP-overproducing (sense-transfected) cells exhibited a marked decrease in the expression of PTH/PTHrP receptor mRNA and PTHrP ligand binding, as well as a corresponding decrease in the PTHrP (1-34)-stimulated cAMP response. By contrast, the antisense-transfected cells showed a marked increase in expression of PTH/PTHrP receptor mRNA and PTHrP (1-34) ligand binding, but a significant increase in the PTHrP (1-34)-stimulated cAMP response was not detected. Using antisense-transfected ROS cells, PTH/PTHrP receptor mRNA expression and 125I-[Tyr36] PTHrP (1-36) binding were downregulated by treatment for 24 h with exogenous PTHrP (1-36), forskolin, or dibutyryl cAMP. The findings extend those of earlier studies showing receptor downregulation by exogenous PTH by indicating that endogenous PTHrP, as well as circulating PTH, may help regulate receptor production; and suggesting that even very low concentrations of the peptide may influence receptor production.

Endogenous parathyroid hormone-related peptide enhances proliferation and inhibits differentiation in the osteoblast-like cell line ROS 17/2.8.

To investigate potential effects of endogenous parathyroid hormone-related peptide (PTHrP) on osteoblast function, ROS 17/2.8 cells were transfected with full-length PTHrP cDNA in a sense or antisense orientation to alter PTHrP production. Compared with vector-transfected control cells, PTHrP-overproducing (sense-transfected) cells showed increased DNA synthesis ([(3)H]-thymidine incorporation) and increased growth (cell number). The extent of apoptosis was compared for the different clones using the terminal deoxynucleotide-mediated dUTP nick-end-labeling assay (TUNEL) and Hoechst staining. No differences in percentages of apoptotic cells were found under basal culture conditions or after 3 days of serum deprivation, which, itself, markedly increased numbers of apoptotic cells. The effect of PTHrP on osteoblast differentiation was assessed by examining two protein markers of differentiation, alkaline phosphatase, and bone morphogenetic protein (BMP)-2. Alkaline phosphatase activity was decreased in sense-transfected cells and increased in antisense-transfected cells, compared with cells transfected with empty vector. PTHrP-overproducing cells also showed decreased numbers of BMP-2-positive cells, whereas antisense-transfected cells showed no difference compared with vector control. The results indicate that: (a) endogenously produced PTHrP can increase growth of these osteoblastic cells by stimulating proliferation while not affecting apoptosis; and (b) the increased cell proliferation produced by PTHrP was accompanied by a reduction in activity or amount of two proteins normally expressed by differentiated osteoblasts.

Single-column purification and bio-characterization of recombinant human parathyroid hormone-related protein (1-139).

Recombinant human parathyroid hormone-related protein (hPTHrP) (1-139) was expressed using the IMPACT T7 (intein-mediated purification with an affinity chitin-binding tag) system, allowing purification of free recombinant peptide in a single chromatographic step. This system utilizes an intein, which is a protein splicing element from the Saccharomyces cerevisiae VMA1 gene. The intein has been modified so that it undergoes a self-cleavage reaction at its N-terminus at low temperatures in the presence of 1,4-dithiothreitol (DTT). The cDNA encoding hPTHrP (1-139) was cloned into the pTYB1 vector to create an in-frame fusion at the N-terminus of the intein gene. The cDNA for the chitin-binding domain from Bacillus circulans is present at the C-terminus of intein for affinity purification of the three-part fusion protein on a chitin column. The recombinant plasmid was transfected into E. coli ER2566 cells and synthesis of the PTHrP fusion protein was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). This system produced pure hPTHrP (1-139) and an N-terminally truncated analogue, hPTHrP (27-139), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot analysis, N-terminal sequence analysis and mass spectroscopy. hPTHrP (1-139) stimulated cAMP accumulation in ROS 17/2.8 osteoblastic bone cells, whereas hPTHrP (27-139) failed to elicit a response. hPTHrP (1-139) also inhibited the growth of the breast cancer cell line MDA-MB-231; the magnitude of the response was comparable with that of synthetic hPTHrP (1-34) and (1-86). Neutralization of endogenous PTHrP and added hPTHrP (1-139) and N-terminal species with an anti-PTHrP antiserum completely abolished the growth inhibitory effects. These results indicate that the added peptides modulate cell growth by acting at the cell surface. Availability of recombinant hPTHrP (1-139) will allow further study of its biological function, as well as its structure.

Stimulatory effect of insulin on calcitonin secretion.

Infusion of insulin directly into thyroid arterial blood perfusing the surgically isolated in situ pig thyroid gland produced an increase in the secretion rate of calcitonin (CT) measured by immunoassay in thyroid venous effluent blood. Insulin in concentrations ranging from approximately 1 to 400 ng/ml produced a maximal stimulation of 4-5 fold. The stimulatory effect of insulin on CT could not be duplicated by infusion of either IGF-I or amylin. Specific binding of radiolabeled insulin was demonstrated using isolated pig thyroid plasma membranes and both rat (6-23) and human (TT) medullary thyroid carcinoma C-cells. Increased CT release was observed from C-cells exposed to a high concentration of insulin. The administration of glucose iv to pigs in order to stimulate secretion of endogenous insulin produced an increase in circulating insulin, which was accompanied by an increase in the secretion of CT. The results show that insulin, delivered directly to the pig thyroid gland, can stimulate CT release. The in vitro binding and secretion studies indicate that C-cells can bind insulin and respond with an increase in CT secretion, and the iv glucose experiments suggest that endogenous insulin is capable of stimulating CT secretion. The findings imply that insulin is capable of acting as a CT secretagogue and suggest that changes in CT secretion may accompany altered states of insulin production such as diabetes or insulin-secreting tumors.

Antitumor effect of positively charged resin in the hamster cheek pouch model.

Following the signal observation that contact with positively charged dextran resin (PCDR) inhibited the growth of cultured mammary (Hs578T and MDA-MB-231), pancreatic (H2T), and myeloma (RR-658) tumor cell lines, studies were developed in the hamster cheek pouch model using hamster H2T pancreatic tumor cells to determine if the antiproliferative effect of PCDR could inhibit tumorigenesis. In these studies, the control population represented groups injected with H2T cells alone or in combination with either neutral or negatively charged resin. When cells (5 x 10(2) to 1 x 10(5)) and PCDR were administered simultaneously, the tumor incidence (percent engraftment) and growth of tumors that already had been established were significantly reduced. When PCDR was injected into already established 1-35-mm2 H2T tumors (engraftment for 21 days = 96%), the resin suppressed the growth of the smallest tumors (< 10 mm2). In none of these trials was the somatic growth of the host hamsters affected. PCDR contact with H2T cells in vitro for 4 days or used to treat growing solid tumors for 72 days significantly reduced cellular ornithine decarboxylase activity. While the mechanism of PCDR action has not been established, the observations have implications for in vivo tumor therapeutic models.

Effect of endogenously produced parathyroid hormone-related peptide on growth of a human hepatoma cell line (Hep G2).

A well differentiated human hepatoma cell line (Hep G2) was used to explore potential roles for PTH-related peptide (PTHrP) as an autocrine/paracrine growth factor. Using Northern analysis or reverse transcription-PCR, Hep G2 cells were found to express messenger RNAs for both PTHrP and the cloned PTH/PTHrP receptor, and the cells exhibited specific binding for [125I]PTHrP(1-36). Hep G2 growth medium was found to contain relatively large amounts of immunoreactive PTHrP (30 vs. 1-2 pM in medium not exposed to cells), and the PTHrP in growth medium (conditioned medium) was shown to contain N-terminal PTHrP biological activity, as assessed by the ability of the medium to stimulate cAMP production in rat osteosarcoma cells (ROS 17/2.8). Conditioned medium produced a dose-dependent severalfold increase in ROS cell cAMP that could be blocked by the PTHrP receptor antagonist [Asn10,Leu11,DTrp12]PTHrP-(7-34). PTHrP in Hep G2 cells also was detected by immunocytochemistry using antiserum to either synthetic PTHrP-(1-34) or recombinant PTHrP-(-5 to 139). Furthermore, these antisera were found to inhibit the ability of PTHrP-(1-34) to stimulate ROS cell cAMP production. When either antiserum (1:800-1:100 dilution) was added to subconfluent Hep G2 cells in medium containing 5% FBS for 3 days, a dose-related 40-50% increase in cell number occurred that could be inhibited by concurrent addition of 10 microM synthetic PTHrP-(1-36). The results show that Hep G2 cells synthesize and secrete both immunoreactive and biologically active PTHrP. As neutralization of PTHrP secreted by these cells by the addition of antiserum to PTHrP resulted in increased cell growth, the findings suggest that PTHrP can function as an autocrine or paracrine growth factor to suppress the growth of these human hepatoma cells.

Alteration of secretion of parathyroid hormone-related peptide and expression of its mRNA in a human hepatoma cell line (HEP G2) treated with agents that affect cell growth.

Previously, using human hepatoma cells (HepG2), we found that immunoneutralization of secreted PTHrP increased cell growth. Here we asked whether PTHrP production was affected by agents that alter growth of Hep G2 cells. Immunoreactive PTHrP in medium and PTHrP mRNA expression were examined. Treatment of cells with 10 µM hydrocortisone or 1 ng/mL TGF-ß1 for 72 h inhibited cell growth by 28±6 and 36±2% and increased PTHrP in medium by 128±10 and 525 ±27%, respectively. The increase in PTHrP produced by both agents was dose-and time-dependent, and the increased PTHrP was accompanied by dose-and time-dependent enhanced expression of PTHrP mRNA. In contrast, 10% fetal bovine serum (FBS) for 72 h increased cell growth by 38±6% (vs serum-free medium) and decreased PTHrP production by 49±4% whereas culture in high glucose (3-4g/L) increased cell growth by 43±1% (vs 1 g/L glucose) and decreased PTHrP by 55±0.4%. Inhibition of PTHrP by both FBS and glucose was dose-dependent; FBS also inhibited PTHrP mRNA. The results show that increased cell growth was associated with decreased PTHrP production, while decreased growth was accompanied by increased PTHrP production. The findings imply that PTHrP may help mediate growth effects of these agents on Hep G2 cells.

Widespread expression of the parathyroid hormone-related peptide and PTH/PTHrP receptor genes in intestinal epithelial cells.

BACKGROUND: This study was designed to determine whether the genes for both parathyroid hormone-related peptide (PTHrP) and its receptor were expressed in close proximity to one another in various regions of the gut and whether they both were evident in two intestinal epithelial cell lines. The findings would test the idea that PTHrP acts as an autocrine or paracrine factor in the gut. EXPERIMENTAL DESIGN: Reverse transcription/PCR and Northern analysis were used to detect mRNA for PTHrP and its receptor in various regions of the normal rat gut, in epithelial cell populations isolated along the villus tip-crypt axis in rat jejunum, and in a rat and a human gastrointestinal epithelial cell line (IEC-6 and LoVo, respectively). Antisera to PTHrP also were used to detect the peptide in rat gastrointestinal tissues or in growth medium from cultured cells. RESULTS: Both PTHrP and PTHrP receptor mRNA were found in all regions of the rat gut, in all cell populations isolated from rat jejunum, and in the rat and human cell lines. Immunoreactive PTHrP in tissue sections was observed in rat jejunal epithelial cells all along the villus but not in crypt cells, and immunoreactive peptide was found in growth medium from the cultured intestinal epithelial cells. CONCLUSIONS: The results show that genes for PTHrP and its receptor are expressed widely in gut epithelia. Expression of the two genes in the same regions of the gut and in the same cell line implies a local autocrine/paracrine action of the peptide. Expression of the peptide in villus epithelium but not in crypt cells suggests a role in differentiating gastrointestinal epithelial cells.

Effects of C-terminal parathyroid hormone-related peptide on osteoblasts.

A C-terminal analog of parathyroid hormone-related peptide (PTHrP), PTHrP(107-139), was found to stimulate cAMP production in three osteoblast cell preparations. The effect was studied most extensively in ROS 17/2.8 cells. The effect was dose-related and comparable in magnitude to that produced by PTHrP(1-34), but potency was lower. The functional significance of the cAMP effect is unknown, but preliminary findings indicated that PTHrP(107-139) also inhibited osteopontin mRNA levels in ROS 17/2.8 cells treated with peptide for 48 h. The results suggest that the carboxy-terminal region of PTHrP may play a role in bone metabolism by influencing osteoblast activity.

Effects of aluminum on rat bone cell populations.

Aluminum (Al) loading is associated with reduced bone formation and osteomalacia in human and certain animal models. However, uncertainty exists as to the cellular effect(s) of Al as both inhibition and stimulation of osteoblast proliferation have been reported. Furthermore, the extent to which Al affects osteoprogenitor cell populations is unknown. To determine the cellular effects of Al in the rat, an animal model in which Al bone disease has been produced, we compared the in vitro effect of 10-50 microns Al on the proliferation and hydroxyproline collagen formation of marrow osteoprogenitor stromal cell populations and perinatal rat calvarial osteoblasts. In subconfluent cultures, Al suppressed proliferation of both marrow fibroblast-like stromal cells and calvarial osteoblasts. In confluent cultures, however, Al selectively stimulated periosteal fibroblast and osteoblast DNA synthesis and collagen (hydroxyproline) production, both in the presence or absence of 1,25-dihydroxyvitamin D. Osteocalcin was not detected in osteoblast-conditioned media or extracellular matrix. These observations suggest that the bone formation defect associated with Al toxicity in growing rats may be a function of impaired patterns of osteoprogenitor/osteoblast proliferation. Furthermore, the Al-stimulated increase in collagen formation is consistent with the development of osteomalacia in Al-toxic humans and animals. The mechanism by which Al stimulated DNA synthesis and collagen production in more mature cultures awaits further study.

Parathyroid hormone-related peptide production and action in a myoepithelial cell line derived from normal human breast.

Physiological roles for PTH-related peptide (PTHrP) appear varied, but remain to be clarified. The peptide is present in large amounts in milk, and PTHrP mRNA has been shown to be present in high amounts in lactating mammary gland. Because PTHrP can cause smooth muscle relaxation, we hypothesized that the peptide might affect the contractility of the breast myoepithelial cell and thereby affect milk ejection. To test this idea, we asked whether PTHrP might affect second messenger responses in a human mammary gland myoepithelial-cell line (Hs578Bst) derived originally from normal breast tissue. To verify the presumed origin of these cells, we also examined the effects of oxytocin. Cells were grown in culture in multiwell plates and exposed to test peptides for 15 min in buffer containing 1 mM isobutylmethylxanthine, and intracellular cAMP was measured by RIA. Both PTHrP-(1-34) and PTH-(1-34) increased cAMP in a dose-related fashion (ED50, 5 nM), with a maximal effect (3-fold) occurring at 100 nM. The ability of PTHrP to stimulate cAMP was inhibited by a 10- to 100-fold molar excess of the specific inhibitors, PTH-(3-34) or PTHrP-(7-34). Inhibitors alone did not alter cAMP. Oxytocin also produced an increase in cAMP, but the effect was inconsistent and occurred only with high doses (0.1-1 microM). Using cells grown on coverslips and loaded with fura-2AM, intracellular Ca2+ was monitored in cells exposed to test peptides. Oxytocin (0.2-20 nM) produced rapid dose-related increases in intracellular Ca2+, with a peak and plateau characteristic of initial mobilization of intracellular Ca2+, followed by entry of extracellular Ca. The plateau was eliminated by the Ca channel antagonist La3+ or by perfusion of cells with Ca-free medium. PTHrP (10-100 nM) altered the intracellular Ca2+ response to oxytocin in 66% of 39 preparations tested. PTHrP inhibited the Ca2+ response when given before oxytocin or transiently decreased the plateau phase of the Ca2+ response when given after oxytocin. Analysis of cellular mRNA using reverse transcription polymerase chain reaction indicated that these cells express the gene for PTHrP, and immunohistochemistry using antiserum to PTHrP revealed positive staining of cells. Measurement of immunoreactive PTHrP in conditioned medium confirmed that these cells can synthesize and secrete the peptide. The finding of a response of this cell line to oxytocin provides functional evidence of their myoepithelial derivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of vasoactive intestinal peptide on adenosine 3',5'-monophosphate, ornithine decarboxylase, and cell growth in a human colon cell line.

Vasoactive intestinal peptide (VIP) is a widely distributed neuropeptide that has been considered a potential regulator of cell growth and differentiation in various tissues, including the gut. To examine this idea, we used a human colon carcinoma cell line (LoVo) as a model system and measured ornithine decarboxylase (ODC), because this is the rate-limiting enzyme for the formation of polyamines, which are thought to be key factors in regulating cell growth. LoVo cells, grown to about 80% confluence in F-12 medium containing 10% fetal bovine serum, were preincubated for 5 h in low serum medium (1% fetal bovine serum in F-12), and ODC activity was determined by measuring 14CO2 liberated from 14C-labeled ornithine. VIP caused a dose-related biphasic change in ODC, with activity increased at 10 pM, maximal (5-fold increase) at 10 nM, and decreased toward basal at 100 nM to 1 microM. Incubation of cells for 6 days with VIP in low serum medium showed similar changes in cell numbers, with growth being increased by doses in the 1 pM to 100 nM range and decreased at higher doses (greater than or equal to 100 nM). Exposure of cells to 5 mM alpha-difluoromethylornithine blocked both the VIP-induced increase in cell number and the VIP-induced increase in ODC activity. Increased ODC mRNA was detected after 2 h of exposure to VIP, a time at which ODC activity peaked after treatment, and the increase in ODC mRNA caused by VIP was dose-dependent. In related experiments LoVo cells were found to have high affinity VIP receptors (Kd = 0.4 nM), as assessed by examination of [125I]VIP binding in the presence of varying concentrations of unlabeled VIP. Studies of intracellular cAMP revealed a dose-related increase in cAMP in response to VIP (ED50 = 11 pM), and the adenylate cyclase activator forskolin increased both ODC activity and ODC mRNA. The findings support the idea that LoVo cells have VIP receptors linked to cAMP which can stimulate cell growth at least in part by increasing ODC synthesis and activity, thereby altering the production of polyamines. The decreased growth and ODC activity observed with high doses of VIP may involve a second messenger other than cAMP.

Effect of transforming growth factor beta on parathyroid hormone receptor binding and cAMP formation in rat osteosarcoma cells.

Transforming growth factor beta (TGF-beta) is now recognized as an important growth regulator and modulator in bone, where it apparently acts in an autocrine or paracrine fashion. In an effort to help elucidate how TGF-beta may interact with parathyroid hormone (PTH) to influence bone turnover, we examined the idea that TGF-beta might alter the number or affinity of PTH receptors in osteoblastic bone cells, PTH receptor binding was assessed in cultured ROS 17/2.8 cells using [125I]PTHrP-(1-34) as labeled ligand. Specific binding to intact cells was measured in the presence of up to 1 microM unlabeled rPTH-(1-34), and cAMP in cell extracts was determined by RIA. Incubation of ROS cells with 2 ng/ml of TGF-beta for the maximally effective time of 3 days increased the number of PTH binding sites (Bmax) by 47 +/- 13%, with no change in the KD (3 nM). TGF-beta also increased the intracellular cAMP response to 0.3 nM rPTH-(1-34) (ED50) by 53 +/- 22%. Both effects were dose dependent, with 1-4 ng/ml of TGF-beta producing maximal effects, and both effects were blocked by the protein synthesis inhibitor cycloheximide (2-5 microM). Since TGF-beta induced comparable increases in both PTH binding and cAMP formation, the findings suggest that TGF-beta can increase the number of functional PTH receptors in cultured ROS 17/2.8 cells. This effect may reflect an action of TGF-beta to slow replication and promote differentiated functions in these cells.

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.

Expression of cholecystokinin forms by medullary thyroid cancer.

We have studied the production and release of cholecystokinin (CCK) forms by rat medullary thyroid cancer (MTC) in vivo. MTC cells were inoculated s.c. into 8 Wag-Rij rats. One month later, after i.v. injection of calcium plasma levels of CCK, calcitonin and tissue contents of gastrin and calcitonin were determined by radioimmunoassay (RIA), immunocytochemistry, and high-pressure liquid chromatography (HPLC). The tumors were passed 4 times to 8 rats in 1-month intervals fasting levels of CCK in control rats were unaffected by calcium stimulation, and in tumor-bearing rats, plasma CCK was elevated in 3 out of 4 passages, falling to normal levels at the end of passage 4. Hypercalcemia had no effect on plasma levels of CCK in tumor-bearing rats, but did stimulate the release of calcitonin in both control and some tumor-bearing rats in later passages. CCK-8 sulfate was found in all 4 tumor passages but not CCK-33/39. We conclude that rat MTC synthesizes and releases CCK-8, but unlike calcitonin, release of CCK appears unresponsive to calcium stimulation.

Stimulation of calcitonin secretion in the pig by calcitonin gene-related peptide.

Pig thyroid glands were surgically isolated in situ and perfused with autologous blood to which was added known concentrations of calcitonin gene-related peptide (alpha CGRP). When thyroids were perfused with measured concentrations of CGRP within the range of 0.6-600 nM, the secretion rate of calcitonin (CT) was stimulated while the release of T3, T4, and somatostatin remained unchanged. Specific binding of 125I-CGRP to pig thyroid plasma membranes was demonstrated, and binding was inhibited by unlabelled CGRP but not by CT or by other peptides unrelated structurally to CGRP. The findings indicate that the pig thyroid gland contains plasma membrane binding sites for CGRP and that CGRP is capable of stimulating the secretion of CT.