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Abstract Background Signal transduction processes mediated by phosphatidyl inositol phosphates affect a broad range of cellular processes such as cell cycle progression, migration and cell survival. The protein kinase AKT is one of the major effectors in this signaling network. Chronic AKT activation contributes to oncogenic transformation and tumor development. Therefore, analogs of phosphatidyl inositol phosphates (PIAs) were designed as new small drugs to block AKT activity for cancer treatment. Here we characterize the biological effects of the PIAs SH-5 and SH-6 in colorectal cancer cell lines. Methods Serum-starved or serum-supplemented human colorectal cancer cell lines SW480, HT29 and HCT116 were exposed to SH-5 and SH-6. AKT activation was determined by western blotting. Cell viability was assessed using a colorimetric XTT-based assay, apoptosis and cell cycle changes were monitored by FACS analysis. The dynamics of cell morphology alterations was evaluated by confocal and time-lapse microscopy. Transcriptional changes due to inhibitor treatment were analyzed using Affymetrix HG-U133A microarrays and RT-PCR. Results While the PIAs clearly reduce AKT phosphorylation in serum starved cells, we did not observe a significant reduction under serum supplemented conditions, giving us the opportunity to analyze AKT independent effects of these compounds. Both inhibitors induce broadly the same morphological alterations, in particular changes in cell shape and formation of intracellular vesicles. Moreover, we observed the induction of binucleated cells specifically in the SW480 cell line. Gene expression analysis revealed transcriptional alterations, which are mostly cell line specific. In accordance to the phenotype we found a gene group associated with mitosis and spindle organization down regulated in SW480 cells, but not in the other cell lines. A bioinformatics analysis using the Connectivity Map linked the gene expression pattern of the inhibitor treated SW480 cells to PKC signaling. Using confocal laser scanning microscopy and time lapse recording we identified a specific defect in the last step of the cytokinesis as responsible for the binucleation. Conclusions The PIAs SH-5 and SH-6 impinge on additional cellular targets apart from AKT in colorectal cancer cells. The effects are mostly cell line specific and have an influence at the outcome of the treatment. In view of potential clinical trials it will be necessary to take these diverse effects into consideration to optimize patient treatment.

The growing epidemic of obesity and metabolic diseases calls for a better understanding of adipocyte biology. The regulation of transcription in adipocytes is particularly important, as it is a target for several therapeutic approaches. Transcriptional outcomes are influenced by both histone modifications and transcription factor binding. Although the epigenetic states and binding sites of several important transcription factors have been profiled in the mouse 3T3-L1 cell line, such data are lacking in human adipocytes. In this study, we identified H3K56 acetylation sites in human adipocytes derived from mesenchymal stem cells. H3K56 is acetylated by CBP and p300, and deacetylated by SIRT1, all are proteins with important roles in diabetes and insulin signaling. We found that while almost half of the genome shows signs of H3K56 acetylation, the highest level of H3K56 acetylation is associated with transcription factors and proteins in the adipokine signaling and Type II Diabetes pathways. In order to discover the transcription factors that recruit acetyltransferases and deacetylases to sites of H3K56 acetylation, we analyzed DNA sequences near H3K56 acetylated regions and found that the E2F recognition sequence was enriched. Using chromatin immunoprecipitation followed by high-throughput sequencing, we confirmed that genes bound by E2F4, as well as those by HSF-1 and C/EBPα, have higher than expected levels of H3K56 acetylation, and that the transcription factor binding sites and acetylation sites are often adjacent but rarely overlap. We also discovered a significant difference between bound targets of C/EBPα in 3T3-L1 and human adipocytes, highlighting the need to construct species-specific epigenetic and transcription factor binding site maps. This is the first genome-wide profile of H3K56 acetylation, E2F4, C/EBPα and HSF-1 binding in human adipocytes, and will serve as an important resource for better understanding adipocyte transcriptional regulation.

Introduction: CD44 is a cell adhesion molecule believed to play a critical role in T cell and monocyte infiltration in the inflammatory process. The reduction of CD44 expression or its ability to properly interact with its key ligand, hyaluronic acid (HA), inhibits migration and subsequent activation of cells within sites of inflammation. CD44-deficient mice exhibit decreased disease in a mouse arthritis model., Methods: Accordingly, we developed PF-03475952, a fully human IgG2 anti-CD44 monoclonal antibody (mAb)., Results: Binding of PF-03475952 to CD44 inhibits binding of HA and induces loss of CD44 from the cell surface. PF-03475952 also passed a series of safety pharmacology assays designed to assess the risk of the mAb to bind Fc gamma receptors, stimulate cytokine release from human whole blood, and stimulate cytokine release from peripheral blood mononuclear cells (PBMC) using plate-bound antibodies. The latter assay was designed specifically to evaluate the risk of cytokine storm that had been observed with TGN1412 (immunostimulatory CD28 superagonist mAb). PF-003475952 exhibits high-affinity binding to both human and cynomolgus monkey CD44, but does not cross-react with rodent CD44. Thus, a rat anti-mouse CD44 mAb was used to demonstrate a dose-dependent decrease of disease in mouse collagen-induced arthritis. Importantly, efficacy was correlated with >50% loss of cell surface CD44 on circulating cells. Loss of CD44 expression on CD3+ lymphocytes was monitored following a single dose of PF-03475952 in cynomolgus monkeys as a pharmacodynamic marker. The recovery of CD44 expression was found to be dose-dependent. PF-03475952 doses of 1, 10, and 100 mg/kg reduced CD44 expression below 50% for 218, 373, and >504 hours, respectively., Conclusion: Targeting of CD44 is a unique mechanism of action in the treatment of inflammatory diseases and is expected to reduce joint damage induced by inflammatory mediators, resulting in disease modification in inflammatory diseases such as rheumatoid arthritis.

Background: Cyclooxygenase-2-specific anti-inflammatory drugs (coxibs) and nonspecific nonsteroidal anti-inflammatory drugs have been shown to inhibit experimental fracture-healing. The present study tested the hypothesis that these effects are reversible after short-term treatment., Methods: With use of a standard model of fracture-healing, identical ED50 dosages of either a nonsteroidal anti-inflammatory drug (ketorolac), a coxib (valdecoxib), or vehicle (control) were orally administered to rats for either seven or twenty-one days and fracture-healing was assessed with biomechanical, histological, and biochemical analyses., Results: When healing was assessed at twenty-one days, the seven-day treatment produced only a trend for a higher rate of nonunion in valdecoxib and ketorolac-treated animals as compared with controls. No differences were observed at thirty-five days. The twenty-one-day treatment produced significantly more nonunions in valdecoxib-treated animals as compared with either ketorolac-treated or control animals (p < 0.05), but these differences disappeared by thirty-five days. The dose-specific inhibition of these drugs on prostaglandin E2 levels and the reversibility of the effects after drug withdrawal were assessed in fracture calluses and showed that ketorolac treatment led to twofold to threefold lower levels of prostaglandin E2 than did valdecoxib. Withdrawal of either drug after six days led to a twofold rebound in these levels by fourteen days. Histological analysis showed delayed remodeling of calcified cartilage and reduced bone formation in association with valdecoxib treatment., Conclusions: Cyclooxygenase-2-specific drugs inhibit fracture-healing more than nonspecific nonsteroidal anti-inflammatory drugs, and the magnitude of the effect is related to the duration of treatment. However, after the discontinuation of treatment, prostaglandin E2 levels are gradually restored and the regain of strength returns to levels similar to control.

Human mesenchymal stem cells (MSCs), bone marrow-derived pluripotent adherent cells of mesenchymal origin can differentiate along the osteogenic, chondrogenic, adipogenic, and tendonogenic lineages. In this report we characterize cytokine and growth factor gene expression by MSCs and investigate the modulation of cytokine expression that occurs during osteogenic and stromal differentiation. MSCs constitutively expressed mRNA for interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), and stem cell factor (SCF). MSCs treated with IL-1alpha upregulated mRNA levels of IL-6, IL-11, and LIF, and began to express detectable levels of granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF). mRNA levels of M-CSF and SCF did not change. MSCs cultured in osteogenic medium differentiated along the osteogenic lineage and downregulated mRNA levels of IL-6, IL-11 and LIF whereas, M-CSF and SCF expression were unchanged and G-CSF and GM-CSF remained undetectable. IL-3 was not detected in MSC culture under any conditions. MSCs precultured in control medium, IL-1alpha, or osteogenic medium maintained similar capacity to support long-term culture initiating cell (LT-CIC). Thus, primary and osteogenic differentiated MSCs produce important hematopoietic cytokines and support hematopoiesis in long-term cultures, suggesting that these cells may provide an excellent ex vivo environment for hematopoiesis during progenitor cell expansion and may be important for in vivo cell therapy.

Mesenchymal stem cells contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose, and marrow stroma. Transduction of mesenchymal stem cells from species other than humans is required for the development of disease models in which mesenchymal stem cells-based gene delivery is evaluated. Attempts to transduce mesenchymal stem cells from some species with amphotropic retroviral vectors were unsuccessful, leading to comparative mesenchymal stem cells transductions with xenotropic and gibbon-ape leukemia virus envelope-pseudotyped retroviral vectors. Human, baboon, canine, and rat mesenchymal stem cells were transduced optimally with amphotropic vector supernatants. In contrast, sheep, goat, and pig mesenchymal stem cells showed highest transduction levels with xenotropic retroviral vector supernatant, and rabbit mesenchymal stem cells were transduced optimally with gibbon-ape-enveloped vectors. Using a myeloablative canine transplantation model and gene-marked canine mesenchymal stem cells, the biodistribution of infused and ex vivo expanded mesenchymal stem cells were examined. The majority of transduced canine mesenchymal stem cells were found in the bone marrow samples. The current study shows the use of mesenchymal stem cells as a delivery vehicle for gene transfer studies, and validates the feasibility of delivering mesenchymal stem cells to the marrow compartment for stromal regeneration after cancer-associated cytotoxic therapies.

Megakaryocytopoiesis and thrombocytopoiesis result from the interactions between hematopoietic progenitor cells, humoral factors, and marrow stromal cells derived from mesenchymal stem cells (MSCs) or MSCs directly. MSCs are self-renewing marrow cells that provide progenitors for osteoblasts, adipocytes, chondrocytes, myocytes, and marrow stromal cells. MSCs are isolated from bone marrow aspirates and are expanded in adherent cell culture using an optimized media preparation. Culture-expanded human MSCs (hMSCs) express a variety of hematopoietic cytokines and growth factors and maintain long-term culture-initiating cells in long-term marrow culture with CD34(+) hematopoietic progenitor cells. Two lines of evidence suggest that hMSCs function in megakaryocyte development. First, hMSCs express messenger RNA for thrombopoietin, a primary regulator for megakaryocytopoiesis and thrombocytopoiesis. Second, adherent hMSC colonies in primary culture are often associated with hematopoietic cell clusters containing CD41(+) megakaryocytes. The physical association between hMSCs and megakaryocytes in marrow was confirmed by experiments in which hMSCs were copurified by immunoselection using an anti-CD41 antibody. To determine whether hMSCs can support megakaryocyte and platelet formation in vitro, we established a coculture system of hMSCs and CD34(+) cells in serum-free media without exogenous cytokines. These cocultures produced clusters of hematopoietic cells atop adherent MSCs. After 7 days, CD41(+) megakaryocyte clusters and pro-platelet networks were observed with pro-platelets increasing in the next 2 weeks. CD41(+) platelets were found in culture medium and expressed CD62P after thrombin treatment. These results suggest that MSCs residing within the megakaryocytic microenvironment in bone marrow provide key signals to stimulate megakaryocyte and platelet production from CD34(+) hematopoietic cells., (Copyright 2000 Wiley-Liss, Inc.)

The nuclear receptor and transcription factor, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), regulates the activity of other transcription factors in the adipogenic differentiation and inflammatory response pathways. We examined the possible function of the PPAR-gamma pathway in osteoclast (Ocl) formation from CD34(+) hematopoietic stem cells (CD34(+) HSCs), using a co-culture system comprised of human mesenchymal stem cells (hMSCs) and CD34(+) HSCs, both derived from bone marrow. Ocl formation in this co-culture system is enhanced by the addition of exogenous osteoprotegerin ligand (OPGL), an essential Ocl differentiation factor, and macrophage-colony stimulating factor (M-CSF). The data indicate that soluble OPGL (sOPGL) and M-CSF stimulate Ocl formation in the co-cultures up to 4-fold compared with CD34(+) HSCs alone treated with sOPGL and M-CSF. CD34(+) HSCs, but not hMSCs, express PPAR-gamma, and 15-deoxy-Delta(12, 14)-prostaglandin-J2 (15d-PG-J2), a PPAR-gamma agonist, completely blocked the effects of sOPGL and M-CSF on Ocl formation and activity. The inhibitory effect of 15d-PG-J2 is specific to the Ocl lineage in both human and mouse models of osteoclastogenesis. Accordingly, parallel experiments demonstrate that sOPGL activates the NF-kappaB pathway within mouse Ocl progenitors, and this effect was abolished by 15d-PG-J2. These data establish a link between PPAR-gamma and OPGL signaling within Ocl progenitors, and support a role for PPAR-gamma pathway in the modulation of osteoclastogenesis.

Mesenchymal stem cells (MSCs) are a population of pluripotent cells within the bone marrow microenvironment defined by their ability to differentiate into cells of the osteogenic, chondrogenic, tendonogenic, adipogenic, and myogenic lineages. We have developed methodologies to isolate and culture-expand MSCs from human bone marrow, and in this study, we examined the MSC's role as a stromal cell precursor capable of supporting hematopoietic differentiation in vitro. We examined the morphology, phenotype, and in vitro function of cultures of MSCs and traditional marrow-derived stromal cells (MDSCs) from the same marrow sample. MSCs are morphologically distinct from MDSC cultures, and flow cytometric analyses show that MSCs are a homogeneous cell population devoid of hematopoietic cells. RT-PCR analysis of cytokine and growth factor mRNA in MSCs and MDSCs revealed a very similar pattern of mRNAs including IL-6, -7, -8, -11, -12, -14, and -15, M-CSF, Flt-3 ligand, and SCF. Steady-state levels of IL-11 and IL-12 mRNA were found to be greater in MSCs. Addition of IL-1alpha induced steady-state levels of G-CSF and GM-CSF mRNA in both cell preparations. In contrast, IL-1alpha induced IL-1alpha and LIF mRNA levels only in MSCs, further emphasizing phenotypic differences between MSCs and MDSCs. In long-term bone marrow culture (LTBMC), MSCs maintained the hematopoietic differentiation of CD34+ hematopoietic progenitor cells. Together, these data suggest that MSCs represent an important cellular component of the bone marrow microenvironment.

Mesenchymal Stem Cells (MSCs) possessing the capacity to differentiate into various cell types such as osteoblasts, chondrocytes, myoblasts, and adipocytes have been previously isolated from the marrow and periosteum of human, murine, lapine, and avian species. This study documents the existence of similar multipotential stem cells in canine marrow. The cells were isolated from marrow aspirates using a modification of techniques previously established for human MSCs (hMSCs), and found to possess similar growth and morphological characteristics, as well as osteochondrogenic potential in vivo and in vitro. On the basis of these results, the multipotential cells that were isolated and culture expanded are considered to be canine MSCs (cMSCs). The occurrence of cMSCs in the marrow was determined to be one per 2.5 x 10(4) nucleated cells. After enrichment of the cMSCs by centrifugation on a Percoll cushion, the cells were cultivated in selected lots of serum. Like the hMSCs, cMSCs grew as colonies in primary culture and on replating, grew as a monolayer culture with very uniform spindle morphology. The population doubling time for these cMSCs was approximately 2 days. The morphology and the growth kinetics of the cMSCs were retained following repeated passaging. The osteogenic phenotype could be induced in the cMSC cultures by the addition of a synthetic glucocorticoid, dexamethasone. In these osteogenic cultures, alkaline phosphatase activity was elevated up to 10-fold, and mineralized matrix production was evident. When cMSCs were loaded onto porous ceramics and implanted in autologous canine or athymic murine hosts, copious amounts of bone and cartilage were formed in the pores of the implants. The MSC-mediated osteogenesis obtained by the implantation of the various MSC-loaded matrix combinations is the first evidence of osteogenesis in a canine model by implantation of culture expanded autologous stem cells. The identification and isolation of cMSCs now makes it feasible to pursue preclinical models of bone and cartilage regeneration in canine hosts.

Osteopetrosis in toothless (tl) rats is characterized by reductions in bone resorption, osteoclasts, and macrophages, resistance to cure by bone marrow transplantation, and skeletal improvement after treatment with colony-stimulating factor-1 (CSF-1). Reductions in skeletal osteocalcin tl rats, together with the recent demonstration of osteocalcin expression in platelets and its possible role in bone turnover, prompted us to examine whether this rat mutation is associated with altered platelet numbers. Our prediction of a thrombocytopenia was confirmed by examination of tl rats, in which a profound reduction (32%) in platelets was accompanied by a significant elevation (62%) in megakaryocytes (MKC) compared to normal littermates. Light and transmission electron microscopy confirmed increases in both number and size of MKC in mutants without morphologic abnormalities of circulating platelets. CSF-1 treatment (10(6) U/48 hours for 10 days) of mutants restored platelet numbers to those found normal littermates and increased osteoclasts and the frequency of MKC in numbers. Preliminary studies of another mutation the rat, osteopetrosis (op), revealed a similar reduction (33%) in platelets. These data demonstrate the coexistence of osteopetrosis and thrombocytopenia in two osteopetrotic rat mutations and an increase in osteoclasts and platelets in one mutation after CSF-1 treatment. Together, these data suggest a potential functional interaction of MKC and osteoclasts in bone turnover.

The gene encoding parathyroid hormone-related protein (PTHrP), an autocrine/paracrine inhibitor of vascular and nonvascular smooth muscle contractility, is regulated by hormonal steroids including estrogens (E2), 1,25-dihydroxy vitamin D (Vit D3) and glucocorticoids. While E2 increases PTHrP gene expression, Vit D3 and glucocorticoids inhibit transcriptional activity of this gene. In the uterus of ovariectomized rats, E2-treatment increases both PTHrP mRNA levels and smooth muscle sensitivity to the action of PTHrP(1-34). To examine the action(s) of Vit D3 and glucocorticoids on these parameters, OVX rats were treated with E2, Vit D3 or the synthetic glucocorticoid, dexamethasone (Dex), alone, or with E2 following a 1 h pretreatment with Vit D3 or Dex. PTHrP and PTH/PTHrP receptor mRNA were measured by blot hybridization analysis of RNA prepared from uteri collected 2, 4 and 24 h after treatment. Uterine horns were used to measure the effect of the steroids on the ability of PTHrP(1-34) to inhibit spontaneous myometrial contraction. When E2, Vit D3 and Dex were given alone, only E2 altered PTHrP mRNA levels in the uterus, however, a 1 h pretreatment with Dex but not Vit D3 markedly diminished this effect of E2. The temporal decline in uterine PTH/PTHrP receptor mRNA levels measured 2 and 4 h after E2 treatment inversely correlated to changes in sensitivity of the tissue to PTHrP(1-34) measured at 24 h after E2 administration. In comparison to E2 alone, treatment with Vit D3 and E2 augmented the uterine responsiveness to PTHrP(1-34) while pretreatment with Dex (1 mg/kg) and E2 decreased this response. These data indicate that in the uterus, Dex opposes the positive effect of E2 on PTHrP gene activity and differentially modulates the action of PTHrP on myometrial tone. Moreover, elevations in the circulating levels of cortisol at term may serve to decrease both the uterine expression of PTHrP and the local action of PTHrP on the myometrium prior to parturition, therefore promoting myometrial contraction associated with labor.

Although mechanical forces regulate bone mass and morphology, little is known about the signals involved in that regulation. External force application increases periosteal bone formation by increasing surface activation and formation rate. In this study, the early tibial periosteal response to external loads was compared between loaded and nonloaded contralateral tibia by examining the results of blot hybridization analyses of total RNA. To study the impact of external load on gene expression, RNA blots were sequentially hybridized to cDNAs encoding the protooncogene c-fos, cytoskeletal protein beta-actin, bone matrix proteins alkaline phosphatase (ALP), osteopontin (Op), and osteocalcin (Oc), and growth factors insulin-like growth factor I (IGF-I) and transforming growth factor-beta (TGF-beta). The rapid yet transient increase in levels of c-fos mRNA seen within 2 hours after load application indirectly suggests that the initial periosteal response to mechanical loading is cell proliferation. This is also supported by the concomitant decline in levels of mRNAs encoding bone matrix proteins ALP, Op, and Oc, which are typically produced by mature osteoblasts. Another early periosteal response to mechanical load appeared to be the rapid induction of growth factor synthesis as TGF-beta and IGF-I mRNA levels were increased in the loaded limb with peak levels being observed 4 hours after loading. These data indicate that the acute periosteal response to external mechanical loading was a change in the pattern of gene expression which may signal cell proliferation. The altered pattern of gene expression observed in the present study supports previous evidence of increased periosteal cell proliferation seen both in vivo and in vitro following mechanical loading.

Osteocalcin (Oc), an abundant gamma-carboxylated protein (mol wt, 5800) of bone matrix, is used as a serum marker of bone turnover because it is considered to be uniquely synthesized by the osteoblast. Our finding of Oc messenger RNA (mRNA) in rat tibial diaphyseal marrow led us to investigate the cellular origins of Oc mRNA in peripheral blood and bone marrow. In anticoagulated blood, Oc mRNA was detected in total RNA prepared from buffy coat cells (BCC). Fractionation of rat and human blood showed that platelets contain Oc mRNA identical to that found in bone cells. In rat bone marrow, Oc mRNA is highly enriched in the platelet-producing megakaryocyte population. Depending upon the RIA used, immunoreactive Oc was either undetectable or present at very low levels in platelets and megakaryocytes, suggesting that synthesis of Oc by these cells may be under strong translational regulation. In addition, Oc levels were higher in serum vs. plasma obtained from the same blood, suggesting that Oc may be released by platelets during blood clotting. Interestingly, the magnitude of this difference was greater in female rats. Injection of 1,25-dihydroxyvitamin D3 dose-dependently increased plasma Oc, but did not cause correlative changes in steady state levels of Oc mRNA in BCC. During rat growth, plasma Oc was maximal, whereas Oc mRNA levels in BBC were low. This relationship was reversed during aging. A correlation between Oc mRNA levels in BCC and rat age suggests a developmental regulation of Oc mRNA levels in platelets. These data indicate that Oc mRNA is not restricted to cells on mineralizing surfaces, but is also found in megakaryocytes and peripheral blood platelets, which possibly contribute to the Oc levels in blood and the regulation of bone turnover.

Parathyroid hormone-related protein shares similarities in sequence and function with the endocrine hormone, parathyroid hormone. However, unlike parathyroid hormone, a product of the parathyroid glands, parathyroid hormone-related protein has a wide distribution in tissues, including the mammary gland. Although during pregnancy the expression of parathyroid hormone-related protein in the mammary gland is low, following birth, protein levels rise sharply in the gland in response to elevations in serum prolactin. Large amounts of parathyroid hormone-related protein are secreted into milk, suggesting a possible role in the neonate. Transient phosphaturia and elevations of parathyroid hormone-related protein in mammary vein plasma support a possible endocrine function for parathyroid hormone-related protein during lactation. Recent evidence suggests a local function for parathyroid hormone-related protein in the lactating mammary gland, and evidence exists that parathyroid hormone-related protein stimulates calcium secretion by the goat mammary gland. Parathyroid hormone-related protein, a putative vasodilator, is produced by the external nutrient vasculature of the mammary gland, and levels within this tissue are regulated during lactation. Infusion of parathyroid hormone-related protein into the ovine mammary artery increases gland blood flow, suggesting a role for the protein in modulation of mammary gland hemodynamics. Regulation of parathyroid hormone-related protein synthesis by the lactating gland, together with the protein's actions on regional blood flow and calcium secretion, support an important function in the mammary gland during lactogenesis.

Parathyroid hormone-related protein (PTHrP) is produced by various normal and neoplastic tissues. Even if the physiological function(s) of PTHrP is unclear, evidence suggests that the protein may participate in the local regulation of smooth muscle contractility. We show here that PTHrP is produced in endothelial cells cultured from human umbilical veins as demonstrated both at the mRNA and protein level. The expression of PTHrP can be upregulated by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate, which is known to stimulate endothelial cell differentiation and angiogenesis in vitro. Unlike smooth muscle cells, the endothelial cells do not express the parathyroid hormone (PTH)/PTHrP receptor mRNA, nor could specific binding of the protein be detected. We therefore suggest that PTHrP produced by endothelial cells acts on smooth muscle cells and may be of importance for the growth and development of new vasculature.

Parathyroid hormone-related protein (PTHrP) gene expression in the pregnant rat uterus has been shown to be dependent on occupancy of the uterus by the fetus. To further test the hypothesis that the synthesis of PTHrP in smooth muscle tissue is regulated by mechanical stretch, we conducted experiments using the rat urinary bladder as a model of an expansible hollow organ. The results indicate that PTHrP mRNA levels do change in response to the stretch of the bladder wall. Under normal conditions PTHrP mRNA levels in the bladder correlated with the urine volume-namely, the extent of bladder distension. When bladders were maintained empty in vivo, PTHrP mRNA levels decreased gradually. Conversely, when bladders were distended by the accumulation of urine, levels of PTHrP mRNA increased dramatically with time. When distension was limited to one-half of the bladder, the increase in PTHrP mRNA was observed only in the distended portion. Histochemical studies performed on distended bladder tissue indicated the presence of PTHrP immunoreactivity in smooth muscle cells. Isolated organ bath studies were used to examine the possible physiological role of PTHrP in smooth muscle tonicity. In vitro responsiveness of bladder muscle strips to exogenous PTHrP was dependent on the in vivo condition of the bladder. In muscle strips obtained from bladders kept empty in vivo, PTHrP-(1-34)-NH2 relaxed carbachol-induced contraction in a dose-dependent manner but failed to relax the contraction in muscle strips from distended bladders that had high endogenous PTHrP expression. These results and the previous findings in the rat uterus suggest a physiological role of PTHrP in bladder smooth muscle function.

PTH-related protein (PTHrP) is found in high concentrations in the milk of various mammals. However, little is known about the regulation of PTHrP production or the physiological role(s) of PTHrP in the mammary glands. To address these questions, we examined in lactating rats 1) the longitudinal changes in PTHrP concentrations in milk and PTHrP mRNA levels in the mammary glands throughout lactation, 2) the effects of the nonsuckling interval on milk PTHrP concentration, and 3) the correlation between PTHrP and calcium concentrations in milk. PTHrP concentrations in milk, measured by RIA and in vitro bioassay, increased with the duration of lactation. The maximal concentrations of PTHrP (observed between days 19-21 of lactation in rats milked serially) were 4.8- to 8.0-fold higher than the concentrations on day 7. PTHrP mRNA levels in the mammary glands also increased during the late stages of lactation. The longitudinal changes in calcium concentrations in milk were small and did not parallel the changes in PTHrP. When pups were removed from the mother for 4-24 h, milk PTHrP decreased while calcium increased in a time-dependent manner. As a whole, calcium concentrations in milk did not correlate with PTHrP throughout lactation. These data suggest that the production and secretion of PTHrP into milk are regulated independently of the other major milk proteins by a factor(s) that changes with progression of lactation and in relation to suckling status.

Earlier studies have shown that lactation-induced bone loss in the rat is both PTH- and vitamin D-independent and have suggested the involvement of another, as yet unidentified, factor(s) in the altered calcium metabolism which accompanies lactation. In the present study, we investigated the possibility that PTH-related protein (PTHrP), which is produced in lactating mammary glands, is a putative calciotropic factor acting systemically during lactation. To test this hypothesis, we examined changes in urinary phosphate and cAMP excretion in relation to suckling since phosphaturia (P-uria) and increased urinary cAMP excretion are sensitive parameters of PTHrP action on the kidney. When lactating rats (separated from their pups overnight) were allowed to suckle pups for 1 h, they showed a marked P-uria which lasted 3-4 h. In most instances, a transient increase in cAMP excretion preceded the P-uria. These effects were not abolished by thyroparathyroidectomy; hence they are not attributable to a transient increase in PTH secretion. Administration of PRL or oxytocin did not induce significant P-uria. When lactating rats were pretreated with anti-PTHrP anti-serum, the suckling-associated P-uria was prolonged and augmented. This prolongation of P-uria was similar to the effects observed when exogenous PTHrP (1-34)amide was administered in the presence of the antiserum. These data support the hypothesis that some of the PTHrP produced in lactating mammary glands may be released systemically during suckling and act in an endocrine manner on target organs such as the kidney.

The phylogenetic conservation of the primary structure of PTH-related protein (PTHrP) supports an important, yet undetermined, role(s) for this molecule in the biology of birds and mammals. As an initial step toward understanding the function of PTHrP in birds, we investigated the expression of PTHrP mRNA in tissues of the egg-laying hen. This analysis revealed that PTHrP mRNA is expressed at various levels in lung, brain, heart, and tissues of the digestive tract, including the proventriculus (secretory stomach), gizzard, and small intestine. In the oviduct tissues of adult birds, PTHrP mRNA was detected in the isthmus (membrane-secreting) and shell gland (calcium-secreting) portions, but not in magnum (albumin secreting) tissue. During oviduct development, high levels of PTHrP mRNA present in the oviducts of the 12-week-old bird suggest a role for PTHrP in oviduct development. Interestingly, as the oviduct matures, relatively high levels of PTHrP mRNA segregate with the distal tissues that ultimately differentiate into the isthmus and shell gland (uterus). To address a possible role for PTHrP in the differentiated function of the shell gland, we followed the expression of PTHrP in the shell gland at different times in the laying cycle and found levels of PTHrP to transiently increase as the egg moves through the oviduct, gradually returning to basal levels in the 15-h calcification period. We localized the cycle-associated fluctuations in PTHrP mRNA levels to the shell gland serosa and smooth muscle layer. Immunoreactive PTHrP was localized to the serosal membrane as well as the smooth muscle layer of serosal arterioles, suggesting that PTHrP may modulate vascular smooth muscle activity. In support of this hypothesis, synthetic chicken PTHrP (1-34)NH2 was found to relax the resting tension of isolated shell gland blood vessels in a dose-dependent manner. Together, these data indicate that the expression of the PTHrP gene in the avian oviduct is both temporally and spatially regulated during the egg-laying cycle and that PTHrP may function as an autocrine/paracrine modulator of shell gland smooth muscle activity of both ductal and vascular origins. The vasorelaxant property of N-terminal fragments of PTHrP supports a role for this molecule in the temporal increase in blood flow to the shell gland during egg calcification.

Administration of 17 beta-estradiol (E2) induces a mitogenic response in the rat uterus. Previous studies have shown that this effect involves the transient activation of c-fos and c-myc expression, followed by significant increases in both DNA synthesis and cell proliferation. Zif268 is a zinc finger-containing, DNA-binding transcription factor that has been implicated in the regulation of cell growth and development and has been shown to be coregulated with c-fos in a number of systems. To determine whether Zif268 is also a target for estrogen regulation, we measured the effects of E2 on Zif268 mRNA expression in the uterus of the ovariectomized rat. In this report we demonstrate that although low levels of Zif268 mRNA expression are detectable in the uteri from ovariectomized control rats, treatment with E2 (4, 40, or 400 micrograms/kg BW) induces a rapid and transient 45- to 50-fold increase in the level of Zif268 mRNA 2 h after E2 treatment. The elevated levels of Zif268 mRNA returned to basal 6 h after hormone treatment. Lower doses of E2 (0.004, 0.04, and 0.4 micrograms/kg) had little or no effect on Zif268 mRNA expression, while higher doses of E2 (4-400 micrograms/kg) resulted in maximal increases in Zif268 expression. Dexamethasone, 5 alpha-dihydrotestosterone, and progesterone had no effect on uterine Zif268 mRNA expression, and the induction of Zif268 by E2 was abolished by pretreating the animals with the RNA synthesis inhibitor actinomycin-D. In addition, stimulation of Zif268 mRNA expression was observed with the short-acting estrogen estriol, suggesting that the response may be specific for estrogenic steroids.(ABSTRACT TRUNCATED AT 250 WORDS)

During pregnancy, elevated levels of PTH-related peptide (PTHrP) persist in the myometrium of the rat uterus. Near term, intrauterine occupancy is correlated with high levels of PTHrP messenger RNA in the gravid horn of the unilaterally pregnant uterus. In nongravid tissue from these same animals the presence of smaller yet significant elevations of PTHrP mRNA suggests that the PTHrP gene also may be regulated by humoral factor(s). To test this hypothesis, we assessed the action of 17 beta-estradiol (E2) on the expression of the PTHrP gene in the uterus of the ovariectomized rat. While low levels of PTHrP mRNA are detected in uteri from ovariectomized rats, a single dose of E2 (4, 40, or 400 micrograms/kg body weight) stimulated a 6- to 8-fold increase in the levels of PTHrP mRNA in the uterus at approximately 2 h after E2 treatment. This increase was transient with levels gradually declining to pretreatment (basal) levels within 24 h. Other steroid hormones tested, including dihydrotestosterone, dexamethasone, and progesterone, failed to stimulate this response. The increase of PTHrP mRNA accumulation required a dose greater than 0.4 micrograms/kg. The magnitude and duration of PTHrP mRNA accumulation were very similar when doses of 40 or 400 micrograms/kg were used. In addition, the stimulation of the PTHrP gene by E2 is neither age dependent nor specific to the rat and is, in part, under transcriptional control. Together, these data indicate that in vivo E2 regulates the levels of PTHrP mRNA in the rat uterus and support a role for E2 in the increased expression of PTHrP mRNA in early gestational tissue, as well as in the nongravid horn of the unilaterally pregnant uterus.

The parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) genes are members of a gene family. Whereas PTH is a classical peptide hormone, mounting evidence suggests that the PTHRP may have predominately local actions. We report here that the PTHRP gene is expressed in rat myometrium, with a major peak in PTHRP mRNA expression occurring in the 48 hr immediately preceding parturition. A similar peak in peptide content was found in tissue extracts by biological and immunological assays, but the PTHRP could not be detected in the peripheral circulation or in uterine vein plasma during late gestation. By in situ hybridization histochemistry, PTHRP mRNA was demonstrated in both the longitudinal and circular layers of smooth muscle but was absent in the endometrium. The rise in myometrial PTHRP mRNA in late gestation was dependent upon intrauterine occupancy; it was greatly reduced or absent in nongravid uterine horns. These findings indicate that the expression of the PTHRP gene in preterm myometrium is under the control of a local stimulus and suggest that the PTHRP may play a paracrine or autocrine role in the uterus during the antepartum period, possibly involving myometrial contractility.

The rat bone/liver/kidney/placenta (BLKP) alkaline phosphatase (ALP) gene is expressed at high level in these particular tissues and at low levels in many other tissues. To study the mechanisms underlying the complex regulation of the rat BLKP ALP expression, we isolated a genomic clone, containing a 10.5-kb insert, which includes the promoter of the BLKP ALP gene with 2 kb of 5' flanking region, its first exon (84bp), and over 7 kb of the first intron. The promoter of the rat BLKP ALP displays features of a "housekeeping" gene promoter: an atypical TATA-box (TTCATAA); 3 potential Spl binding sites; high GC content (82% in positions-134 to -14); and a high CpG to GpC ratio (60:89 in the 0.85 kb promoter region), indicating an abundance of potential methylation sites. Likewise, transient transfection of CAT fusion genes into ROS 17/2.8 osteoblast-like cells reveals weak expression from the promoter and proximal 5' flanking sequences, which can be elevated by an SV40E enhancer. The homologous human bone/liver/kidney (BLK) ALP promoter, which demonstrates a similar combination of tissue-specific and housekeeping characteristics, shares close similarity (184 bp of 79% similarity excluding gaps) with the rat BLKP ALP promoter. The human placental ALP is encoded by a separate gene and its promoter, on the other hand lacks significant similarity to the rat BLKP ALP promoter despite their common expression in the placenta. This lack of similarity appears to reflect the close evolutionary relationship of the human placental ALP gene to the intestinal ALP gene. Significant sequence similarity was found between the rat and human BLK/BLKP ALP promoters and the human and mouse adenine deaminase promoters, and together they may represent a class of dual-function promoters, allowing both constitutive low-level, and tissue-specific higher levels of expression. A pentanucleotide with the consensus sequence 5'-GGCTC-3' is present in these promoters and in the promoters for the human fibronectin and the human alpha 1(II) procollagen genes in the region of maximal similarity with the rat BLKP ALP promoter, and in the vicinity of the Sp1-binding sites.

This investigation addresses a theoretical concept of tumor pathogenesis proposed over 40 years ago, namely that malignancy-associated hypercalcemia can result from endocrine secretion by tumors of a PTH-like factor. These studies demonstrate that a fragment of hHCF alone, without added or tumor-secreted cofactors or hormones, can produce hypercalcemia and other biochemical abnormalities associated with HHM. The hypercalcemia can be generated by hHCF-(1-34)NH2 action on bone, although kidney and gut could contribute to the HHM syndrome when it occurs naturally. No other tumor-secreted peptide displays this biological profile. These studies establish one (PTH-like) mechanism by which human tumors could produce hypercalcemia. Furthermore, the finding that hHCF-(1-34)NH2 is more potent than PTH in some systems is of considerable interest for the future design of hormone analogs. A broad spectrum of biological properties of hHCF-(1-34)NH2, including production of components of the HHM syndrome, can be inhibited by a PTH antagonist. Because [Tyr-34]bPTH-(7-34)NH2 selectively and competitively occupies PTH receptors, our studies demonstrate formally that hHCF-(1-34)NH2 mediates some (and perhaps all) of its actions via receptors conventionally regarded as intended for interaction with PTH, but which actually may be present to allow for expression of bioactivity of both secreted proteins. Although some structural homology is shared by the two hormones and many contribute to interaction with receptors, the disparity in structure, especially within the 1-34 domains responsible for bioactivity in both hormones, is more pronounced. The similarity in biological profiles despite structural differences between hHCF and PTH is emphasized by the inhibitory action of [Tyr-34]bPTH-(7-34)NH2 against the tumor peptide even in the absence of much of the homologous region in the PTH antagonist. This investigation provides impetus for designing more potent antagonists, which must now be regarded more appropriately as inhibitors of both PTH and hHCF. Such antagonists may best be generated from hybrid structures of the two hormones. In any case, these studies establish a promising new approach to therapy of tumor-associated hypercalcemia.

A survey of rat tissues by RNA analysis, aimed at uncovering the physiological function of the parathyroid hormone-like peptide (PTH-LP) associated with hypercalcemia of malignancy, revealed the presence of a 1.5-kilobase messenger RNA encoding this peptide in lactating mammary glands. PTH-LP messenger RNA is expressed in mammary tissue only during lactation; it appears and disappears rapidly (2 to 4 hours) as a function of the sucking stimulus. The identity of this messenger RNA was confirmed by cloning the rat PTH-LP complementary DNA, which predicts a peptide with strong similarity to the human homolog. Moreover, extracts from lactating mammary tissue stimulated parathyroid hormone-dependent adenylate cyclase. These findings suggest that PTH-LP plays a physiological role in lactation, possibly as a hormone for the mobilization or transfer (or both) of calcium to the milk.

This study examines the osteoblastic properties of the established human osteosarcoma cell line Saos-2. Saos-2 cells inoculated into diffusion chambers, which were implanted i.p. into nude mice, produced mineralized matrix in 4 of 6 chambers at 8 weeks. In 5 of 6 chambers there was a strong positive alkaline phosphatase reaction. In culture the alkaline phosphatase levels increased with time and cell density, reaching very high levels at confluence: 4-7 mumol/mg protein/min. The cells show a sensitive adenylate cyclase response to parathyroid hormone, 50% effective dose = 2.8 nM, which increases with cell density and is further raised by dexamethasone treatment. They also exhibit typical binding of 1-25-dihydroxyvitamin D3 to 3.2S receptor protein with an apparent Kd of 0.21 nM; the numbers of sites per cell were 3,300 at 50,000 cells/cm2 and 1,800 at 280,000 cells/cm2. The presence of osteonectin was visualized with a monoclonal antibody which revealed a reticular pattern on the cell surface. Osteonectin was also detected in the medium by Western blots, migrating at around Mr 40,000 in nonreduced gels and Mr 44,000 in reduced gels. The Saos-2 cells thus possess several osteoblastic features and could be useful as a permanent line of human osteoblast-like cells and as a source of bone-related molecules.

Hepatic poly(A+) RNA from rats induced for stearyl-CoA desaturase was used for primer-extension of cDNA coding for stearyl-CoA desaturase. Previously, Northern blot analysis showed that translatable desaturase mRNA is 4,900 nucleotides in length (Thiede, M. A., and Strittmatter, P. (1985) J. Biol. Chem. 260, 14459-14463). Six overlapping cDNAs, ranging from 850 to 1450 bases, were used to compile the 4,689-nucleotide sequence. The cDNA includes a 1,074-base open reading frame coding for 358 amino acids, corresponding to a molecular mass of 41,400 daltons. Positive identification of this open reading frame was accomplished by matching the amino acid sequence of both amino-terminal and cyanogen bromide peptides of the purified enzyme with regions of the sequence deduced from the cDNA. Amino acid composition data from the cDNA compares well with that from the desaturase. The protein contains 62% hydrophobic amino acids. An interesting feature of this mRNA is the 3,500-base 3' noncoding region, which has been localized on a single 3' exon by Southern blot analysis.

Alkaline phosphatase [ALP; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] is a ubiquitous enzyme of unknown function expressed at high levels in cells of mineralizing tissues. To study the structure, function, and expression of ALP, a full-length cDNA of rat ALP (2415 bases) was isolated from a ROS 17/2.8 osteosarcoma cell lambda gt10 cDNA library. The predicted amino acid sequence spans 524 residues and includes an N-terminal signal peptide of 17 amino acids, the phosphohydrolase active site, a rather hydrophilic backbone with five potential N-glycosylation sites, and a short hydrophobic C-terminal sequence. ALP negative CHO cells transfected with an expression vector containing the ALP coding sequences express ALP. The rat bone, liver, and kidney ALP shows remarkable 90% homology with the corresponding human enzyme, the most divergent region being the C-terminal hydrophobic domain through which the enzyme may be anchored to the plasma membrane. The rat ALP also shows 50% homology with the human placental and intestinal ALP and 25% homology with the Escherichia coli ALP. The amino acids involved in catalysis show nearly complete homology among all known ALP sequences, suggesting that these enzymes evolved from a common ancestral gene. The rat ALP cDNA pRAP 54, used as a hybridization probe in RNA blot analysis of several tissues that express ALP, revealed the presence of an ALP mRNA of approximately equal to 2500 bases. Furthermore, hybridization patterns derived from Southern blot analysis of rat chromosomal DNA offered molecular evidence that the ALP expressed in ROS 17/2.8 osteosarcoma and various rat tissues, excluding the intestine, is the product of the same single copy gene.

A clonal cell line (Saos-2/B-10) derived from human osteosarcoma Saos-2 cells had the same osteoblastic characteristics as the mother line, but lacked sensitivity to parathyroid hormone (PTH) at early passages. At later passages (greater than 70) the cells became very sensitive to PTH (0.1 nmol/l). The absence of PTH-stimulatable adenylate cyclase correlated with the secretion of an adenylate cyclase-stimulatory activity which had the properties of the recently characterized PTH-like peptide (PTH-LP). This activity was inhibited by the PTH antagonist [8norleucyl,18norleucyl,34tyrosinyl]bovine PTH-(3-34)amide and could be neutralized by an antiserum raised against the synthetic PTH-LP-(1-34). Hybridization with a human PTH-LP cDNA showed that these cells produce two PTH-LP mRNAs of approximately 1.5 and 1.8 kb. The production of PTH-LP was stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 150 nmol/l) and epidermal growth factor (EGF; 10 ng/ml). The increased accumulation of PTH-LP in conditioned media in response to TPA was seen after 1 h and levelled off at 6 h. In contrast, EGF stimulation was lower at 3 and 6 h but continued for 24 h. Both agents increased PTH-LP mRNA levels in Saos-2/B-10 cells. A TPA analogue which does not stimulate protein kinase C had no effect on PTH-LP production. Cycloheximide blocked the stimulatory effect of both TPA and EGF and the TPA effect was blocked by actinomycin D, suggesting transcriptional control. The regulation of PTH-LP by these agents may offer clues regarding the association of this protein with malignancy.

During lactation, a dramatic rise in serum PRL stimulates milk production, resulting in a substantial rise in calcium mobilization from gut and bone. We found that the production of a newly characterized calcium-mobilizing PTH-like peptide (PTH-LP) by mammary tissue was tightly linked to lactation, suggesting a possible role for PRL in the expression of PTH-LP. Here it is shown that suckling results in both an elevation in serum PRL and the appearance of PTH-LP mRNA in mammary tissue. Bromocriptine, a potent inhibitor of PRL secretion, blocked the suckling-associated rise in serum PRL and the subsequent induction of PTH-LP mRNA in mammary gland. Furthermore, injection of PRL dramatically induced PTH-LP mRNA in unsuckled puerperal glands, but not in glands on day 21 of pregnancy. Thus, the correlation between serum levels of PRL and the expression of PTH-LP mRNA in mammary tissue extends the role of PRL in milk production and suggests a possible mechanism for the PRL effects on calcium metabolism.

Two clones containing inserts in pBR322 that together include the entire 1074-base open reading frame coding for the 358 amino acids of rat liver stearyl-CoA desaturase have been used to construct expression vectors for residues 3-358 and 27-358 fused to the first 6 residues of beta-galactosidase and several amino acids of the multiple cloning site of pUC8. Growth of transformed Escherichia coli under conditions for suppression of the lac promoter, followed by subsequent induction of these cultures results in the synthesis of higher levels of desaturase proteins than those found in induced rat liver. The proteins are almost completely associated with the membrane fraction of cell homogenates. Posttranslational iron insertion into the apoproteins, either in vitro with membrane preparations or by iron addition during induction, results in the formation of active holoenzyme which can be reconstituted with NADH cytochrome b5 reductase and cytochrome b5 to form an active stearyl-CoA desaturase system. The deletion of the first 26 amino-terminal amino acid residues does not affect either enzyme activity or membrane binding. Therefore, the unusual sequence of 11 residues containing 10 amino acids with hydroxyl groups plays no apparent significant role in either protein insertion into membranes or iron chelation. Since the protein product for residues 3-358 is processed even further to delete the initial 33 amino-terminal residues, the limiting polypeptide primary structure required for an active membrane-bound catalyst is even smaller than this initial deletion mutation indicates.

Poly(A+) RNA isolated from livers of rats induced for stearyl-CoA desaturase contains elevated levels of mRNA for this enzyme which is translated in a rabbit reticulocyte system. The protein is immunologically and by peptide fingerprinting following Staphylococcus aureus V8 protease digestion identical to the isolated enzyme and, therefore, not synthesized in a detectable larger precursor form. The desaturase mRNA is selectively translated on free cytoplasmic polysomes from rat liver and represents at least a 40-fold increase in translatable mRNA in livers of induced animals. Northern blot analysis, using a cDNA probe complementary to rat liver desaturase mRNA, demonstrated that the desaturase is encoded by a 4900-base mRNA which is elevated approximately 50-fold in induced liver.

A peptide secreted by tumors associated with the clinical syndrome of humoral hypercalcemia of malignancy was recently purified from human renal carcinoma cell line 786-0. The N-terminal amino acid sequence of this peptide has considerable similarity with those of parathyroid hormone (PTH) and of peptides isolated from human breast and lung carcinoma (cell line BEN). In this study we obtained the nucleotide sequence of a 1595-base cDNA complementary to mRNA encoding the PTH-like peptide produced by 786-0 cells. The cDNA contains an open reading frame encoding a leader sequence of 36 amino acids and a 139-residue peptide, in which 8 of the first 13 residues are identical to the N terminus of PTH. Through the first 828 bases the sequence of this cDNA is identical with one recently isolated from a BEN cell cDNA library; however, beginning with base 829 the sequences diverge, shortening the open reading frame by 2 amino acids. Differential RNA blot analysis revealed that 786-0 cells express two major PTH-like peptide mRNAs with different 3' untranslated sequences, one of which hybridizes with the presently described sequence and the other one with that reported for the BEN cell PTH-like peptide cDNA. Primer-extension analysis of 786-0 poly(A)+ RNA together with Southern blot analysis of human DNA confirmed the presence of a single-copy gene coding for multiple mRNAs through alternate splicing. In addition, the 3' untranslated sequence of the cDNA described here has significant similarity to the c-myc protooncogene.

This study examines the use of alkaline phosphatase (AP) as a reporter enzyme. We constructed a plasmid containing the cDNA which encodes the bone/liver/kidney rat AP under the control of the simian virus 40 (SV40) early promoter and used it to transfect Chinese hamster ovary, SV40-transformed African Green Monkey kidney 7, and rat osteosarcoma 25/1 mammalian cells. AP activity in these cells, measured three days later, was 40-400-fold above background. When AP and chloramphenicol acetyltransferase (CAT) plasmids were cotransfected, the detection of AP activity by a simple spectrophotometric assay was at least as sensitive as the detection of CAT activity using a radioactive substrate. Moreover, since mammalian AP is a membrane-bound ectoenzyme, transfected cells can be visualized by histochemical staining. This approach was used to estimate transfection efficiency. The convenient methods for AP detection should make it a useful reporter enzyme.