Phase 3 study of recombinant von Willebrand factor in patients with severe von Willebrand disease who are undergoing elective surgery.

Essentials Recombinant von Willebrand factor (rVWF) is effective in von Willebrand disease (VWD). A phase 3 study of rVWF, with/without recombinant factor VIII (rFVIII) before surgery in VWD. Overall rVWF's efficacy was rated excellent/good; rVWF was administered alone in most patients. rVWF was well-tolerated and hemostasis was achieved in patients with severe VWD undergoing surgery. SUMMARY: Background Recombinant von Willebrand factor (rVWF) has demonstrated efficacy for on-demand treatment of bleeding in severe von Willebrand disease (VWD), warranting evaluation in the surgical setting. Objectives This study (NCT02283268) evaluated the hemostatic efficacy/safety profile of rVWF, with/without recombinant factor VIII (rFVIII), in patients with severe VWD undergoing surgery. Patients/Methods Patients received rVWF 40-60 IU kg-1 , VWF ristocetin cofactor activity was measured 12-24 h before surgery. If endogenous FVIII activity (FVIII:C) target levels were achieved 3 h before surgery, rVWF was administered alone 1 h before surgery; rVWF was co-administered with rFVIII if target endogenous FVIII levels were not achieved. rVWF was infused postoperatively to maintain target trough levels. Overall and intraoperative hemostatic efficacy, the pharmacodynamics of rVWF administration and the incidence of adverse events (AEs) were assessed. Results All patients treated with rVWF for major (n = 10), minor (n = 4) and oral (n = 1) surgery had overall and intraoperative hemostatic efficacy ratings of excellent (73.3% and 86.7%) or good (26.7% and 13.3%). Most rVWF infusions (89.4%) were administered alone, resulting in hemostatically effective levels of endogenous FVIII within 6 h, which were sustained for 72-96 h; 70% (n = 7/10) of major surgeries were performed without rFVIII co-administration. Six patients reported 12 treatment-emergent AEs. Two patients each had one serious AE: diverticulitis (not treatment related) and deep vein thrombosis (sponsor-assessed as possibly treatment related). No severe allergic reactions or inhibitory antibodies were reported. Conclusions These data support the efficacy and safety profile of rVWF in patients with severe VWD undergoing elective surgery.

Erythropoietin: new approaches to improved molecular designs and therapeutic alternatives.

Erythropoietin (Epo) is a glycoprotein hormone that is the prime regulator of erythropoiesis. Recombinant Epo is a highly effective pharmaceutical used to correct anemias associated with renal insufficiency, cancer and other diseases. Efforts to increase its efficacy in vivo by manipulating the protein's structure have met with some success, and novel Epo-like agents are in development. Additionally, efforts to create Epo mimetic agents are underway, as is the design of agents to increase endogenous production. Because Epo has tissue protective actions outside of erythropoiesis, other designs have focused on producing erythropoietically inactive molecules that still retain extra-hematopoietic activity. The demonstration that Epo can trigger signaling in some cancer cells with, potentially, adverse effects on patient health has raised warning signs in the medical community and has gained the attention of regulatory authorities.

Erythropoietin activates two distinct signaling pathways required for the initiation and the elongation of c-myc.

Erythropoietin (Epo) stimulation of erythroid cells results in the activation of several kinases and a rapid induction of c-myc expression. Protein kinase C is necessary for Epo up-regulation of c-myc by promoting elongation at the 3'-end of exon 1. PKCepsilon mediates this signal. We now show that Epo triggers two signaling pathways to c-myc. Epo rapidly up-regulated Myc protein in BaF3-EpoR cells. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 blocked Myc up-regulation in a concentration-dependent manner but had no effect on the Epo-induced phosphorylation of ERK1 and ERK2. LY294002 also had no effect on Epo up-regulation of c-fos. MEK1 inhibitor PD98059 blocked both the c-myc and the c-fos responses to Epo. PD98059 and the PKC inhibitor H7 also blocked the phosphorylation of ERK1 and ERK2. PD98059 but not LY294002 inhibited Epo induction of ERK1 and ERK2 phosphorylation in normal erythroid cells. LY294002 blocked transcription of c-myc at exon 1. PD98059 had no effect on transcription from exon 1 but, rather, blocked Epo-induced c-myc elongation at the 3'-end of exon 1. These results identify two Epo signaling pathways to c-myc, one of which is PI3K-dependent operating on transcriptional initiation, whereas the other is mitogen-activated protein kinase-dependent operating on elongation.

Erythroid cell growth and differentiation in vitro in the simulated microgravity environment of the NASA rotating wall vessel bioreactor.

Prolonged exposure of humans and experimental animals to the altered gravitational conditions of space flight has adverse effects on the lymphoid and erythroid hematopoietic systems. Although some information is available regarding the cellular and molecular changes in lymphocytes exposed to microgravity, little is known about the erythroid cellular changes that may underlie the reduction in erythropoiesis and resultant anemia. We now report a reduction in erythroid growth and a profound inhibition of erythropoietin (Epo)-induced differentiation in a ground-based simulated microgravity model system. Rauscher murine erythroleukemia cells were grown either in tissue culture vessels at 1 x g or in the simulated microgravity environment of the NASA-designed rotating wall vessel (RWV) bioreactor. Logarithmic growth was observed under both conditions; however, the doubling time in simulated microgravity was only one-half of that seen at 1 x g. No difference in apoptosis was detected. Induction with Epo at the initiation of the culture resulted in differentiation of approximately 25% of the cells at 1 x g, consistent with our previous observations. In contrast, induction with Epo at the initiation of simulated microgravity resulted in only one-half of this degree of differentiation. Significantly, the growth of cells in simulated microgravity for 24 h prior to Epo induction inhibited the differentiation almost completely. The results suggest that the NASA RWV bioreactor may serve as a suitable ground-based microgravity simulator to model the cellular and molecular changes in erythroid cells observed in true microgravity.

Novel interaction between the transcription factor CHOP (GADD153) and the ribosomal protein FTE/S3a modulates erythropoiesis.

The transcription factor CHOP (GADD153) heterodimerizes with other C/EBP family members, especially C/EBPbeta, thus preventing their homodimerization and binding to DNA sequences specific for the homodimers. Some CHOP-C/EBP heterodimers apparently bind to alternative DNA sequence and thereby regulate the transcription of other genes. Recently, we demonstrated that CHOP is up-regulated during certain stages of erythroid differentiation and that ectopic overexpression of CHOP enhances this process (Coutts, M., Cui, K., Davis, K. L., Keutzer, J. C., and Sytkowski, A. J. (1999) Blood 93, 3369-3378). In the present study, we report that CHOP also interacts with another non-C/EBP protein designated v-fos transformation effector (FTE) (Kho, C. J., and Zarbl, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2200-2204), which is identical to ribosomal protein S3a (Metspalu, A., Rebane, A., Hoth, S., Pooga, M., Stahl, J. , and Kruppa, J. (1992) Gene (Amst.) 119, 313-316). Bacterially expressed His-CHOP and in vitro translated (35)S-labeled FTE/S3a-Gal4 fusion protein co-immunoprecipitated using anti-CHOP antibodies, and both anti-CHOP and anti-FTE/S3a antibodies co-immunoprecipitated CHOP and FTE/S3a from lysates of Rauscher murine erythroleukemia cells overexpressing both proteins. The in vivo interaction of CHOP and FTE/S3a was also demonstrated in cells overexpressing FTE/S3a but with endogenous expression levels of CHOP. Western blot analysis demonstrated co-localization of CHOP and FTE/S3a in both the cytosol and the nuclei of non-transfected cells. Overexpression of FTE/S3a inhibited differentiation of Rauscher cells induced either by erythropoietin or by dimethyl sulfoxide. This inhibition was reversed partially by simultaneous overexpression of CHOP or of antisense fte/S3a. FTE/S3a appears to be a bifunctional ribosomal protein that regulates CHOP and, hence, C/EBP function during erythropoiesis.

An erythropoietin fusion protein comprised of identical repeating domains exhibits enhanced biological properties.

The hematopoietic growth factor erythropoietin (Epo) initiates its intracellular signaling cascade by binding to and inducing the homodimerization of two identical receptor molecules. We have now constructed and expressed in COS cells a cDNA encoding a fusion protein consisting of two complete human Epo domains linked in tandem by a 17-amino acid flexible peptide. On SDS-polyacrylamide gel electrophoresis, the Epo-Epo fusion protein migrated as a broad band with an average apparent molecular mass of 76 kDa, slightly more than twice the average apparent molecular mass of Epo, 37 kDa. Enzymatic N-deglycosylation resulted in an Epo-Epo species that migrated on SDS-polyacrylamide gel electrophoresis as a narrow band with an average apparent molecular mass of 39 kDa. The specific activity of the Epo-Epo fusion protein in vitro (1,007 IU/microgram; 76 IU/pmol) was significantly greater than that of Epo (352 IU/microgram; 13 IU/pmol). Moreover, secretion of Epo-Epo by COS cells was 8-fold greater than that of Epo. Subcutaneous administration of a single dose of Epo-Epo to mice resulted in a significant increase in red blood cell production within 7 days. In contrast, administration of an equivalent dose of conventional recombinant Epo was without effect. The pharmacokinetic behavior of Epo-Epo differed significantly from that of Epo. The results suggest that Epo-Epo may have important biological and therapeutic advantages.

Regulated expression and functional role of the transcription factor CHOP (GADD153) in erythroid growth and differentiation.

The hematopoietic growth factor erythropoietin (Epo) triggers changes in the expression of genes that encode important regulators of erythroid cell growth and differentiation. We now report that Epo markedly upregulates chop (gadd153) expression and that this transcription factor plays a role in erythropoiesis. Using a differential hybridization assay, we isolated a full-length cDNA of chop as an Epo upregulated gene in Rauscher murine erythroleukemia cells. RNase protection assays demonstrated that Epo or dimethyl sulfoxide induction increased steady-state mRNA levels 10- to 20-fold after 24 to 48 hours. Western blot analysis confirmed a marked increase in CHOP protein. Among the other c/ebp family members, only c/ebp beta was also upregulated during erythroid differentiation. Among normal hematopoietic cells examined, steady-state mRNA levels were highest in erythroid cells, with levels peaking during terminal differentiation. Transient overexpression of chop in Rauscher cells resulted in a significant increase in Epo- or dimethyl sulfoxide (DMSO)-induced hemoglobinization, further linking chop upregulation to erythroid differentiation. Artificial downregulation of chop in normal murine bone marrow cells with antisense oligodeoxynucleotides inhibited colony-forming unit-erythroid (CFU-E)-derived colony growth in a concentration-dependent manner. Burst-forming unit-erythroid (BFU-E)-derived colony growth was not affected. Using a Far Western type of analysis, we detected several potential CHOP binding partners among the nuclear proteins of Rauscher cells. Importantly, the number and relative abundance of these proteins changed with differentiation. The results strongly suggest that CHOP plays a role in erythropoiesis, possibly through interactions with both C/EBP and non-C/EBP family members.

Troponin I is present in human cartilage and inhibits angiogenesis.

Cartilage is an avascular and relatively tumor-resistant tissue. Work from a number of laboratories, including our own, has demonstrated that cartilage is an enriched source of endogenous inhibitors of angiogenesis. In the course of a study designed to identify novel cartilage-derived inhibitors of new capillary growth, we have purified an inhibitory protein that was identified by peptide microsequencing and protein database analysis as troponin I (TnI). TnI is a subunit of the troponin complex (troponin-C and troponin-T being the other two), which, along with tropomyosin, is responsible for the calcium-dependent regulation of striated muscle contraction; independently, TnI is capable of inhibiting actomyosin ATPase. Because troponin has never previously been reported to be present in cartilage, we have cloned and expressed the cDNA of human cartilage TnI, purified this protein to apparent homogeneity, and demonstrated that it is a potent and specific inhibitor of angiogenesis in vivo and in vitro, as well as of tumor metastasis in vivo.

Isolation of differentially expressed genes by cloning transcriptionally active DNA fragments.

During studies of erythroid cell growth and differentiation induced by erythropoietin (Epo), we developed a method that allows the identification and isolation of genes based upon their transcriptional activity. Transcriptionally active genomic DNA fragments from Epo-treated cells and control cells are purified from inactive chromatin using mercury affinity chromatography, based on the mechanism that the thiol groups of histone H3 on transcriptionally active chromatin are exposed to the solvent and therefore are easily accessible. Using the purified genomic DNA fragments from the two populations of cells, a subtractive hybridization strategy is used to isolate and clone genes that are differentially expressed in the absence or in the presence of Epo.

Identification of TAXREB107 as an erythropoietin early response gene.

The protooncogenes c-myc and c-myb are erythropoietin (Epo)-regulated early response genes. They appear to play distinct roles in the growth- and differentiation-inducing signals of the hormone. Using a subtractive hybridization strategy, we have identified the murine homolog of the TAX response element-binding protein TAXREB107 as an Epo early response gene in Rauscher murine erythroleukemia cells. This was confirmed by Northern blot analyses, which showed a fourfold increase in TAXREB107 mRNA after 1 hr of erythropoietin treatment. After 3 hr the transcript had decreased to approximately twofold above control levels. Inhibition of this induction with antisense oligodeoxynucleotides increased Epo-induced hemoglobinization 2.5-fold. The results implicate TAXREB107 in erythropoiesis and support the hypothesis that the TAXREB proteins have functions outside the context of HTLV-I infection.

Identification of genes expressed differentially by LNCaP or PC-3 prostate cancer cell lines.

The transition from androgen-dependent to aggressive androgen-independent growth is an important development in prostate cancer, and the identification of genes expressed differentially between these two phenotypes could provide new therapeutic targets as well as prognostic markers. Using a subtractive approach designated linker capture subtraction, we have now isolated several genes expressed differentially between the human prostate carcinoma cell lines LNCaP and PC-3, which are androgen-dependent and androgen-independent, respectively. In addition to 10 known genes that may be involved in signal transduction, tumor growth, tumor invasion, and metastasis, 3 novel genes were isolated. One of these, designated NPG-1, was expressed by the majority of primary tumors examined. Moreover, the degree of NPG-1 expressed correlated with an aggressive histopathology.

Differential expression and androgen regulation of the human selenium-binding protein gene hSP56 in prostate cancer cells.

Low levels of dietary selenium are associated with increased risk of malignancy of several organs, including the prostate. Using a subtractive approach called linker capture subtraction, we have found that the human selenium-binding protein gene hSP56 is differentially expressed by the relatively slow-growing, androgen-sensitive prostate cancer cell line LNCaP but not by the more rapidly growing androgen-insensitive lines PC-3 and DU145. We confirmed this differential expression by Northern blot analysis. Importantly, hSP56 expression by LNCaP cells was reversibly down-regulated by exogenous androgen in a concentration-dependent manner. Marked differences in steady-state hSP56 mRNA levels were found in a variety of normal and neoplastic human cells that were examined. hSP56 expression was especially high in normal tissues that appear to benefit from the cancer-protective action of dietary selenium and was low in many neoplastic cells. The results suggest that hSP56 may play a role in determining the neoplastic phenotype.

Human erythropoietin dimers with markedly enhanced in vivo activity.

Human erythropoietin, a widely used and important therapeutic glycoprotein, has a relatively short plasma half-life due to clearance by glomerular filtration as well as by other mechanisms. We hypothesized that an erythropoietin species with a larger molecular size would exhibit an increased plasma half-life and, potentially, an enhanced biological activity. We now report the production of biologically active erythropoietin dimers and trimers by chemical crosslinking of the conventional monomeric form. We imparted free sulfhydryl residues to a pool of erythropoietin monomer by chemical modification. A second pool was reacted with another modifying reagent to yield monomer with maleimido groups. Upon mixing these two pools, covalently linked dimers and trimers were formed that were biologically active in vitro. The plasma half-life of erythropoietin dimers in rabbits was >24 h compared with 4 h for the monomers. Importantly, erythropoietin dimers were biologically active in vivo as shown by their ability to increase the hematocrits of mice when injected subcutaneously. In addition, the dimers exhibited >26-fold higher activity in vivo than did the monomers and were very effective after only one dose. Dimeric and other oligomeric forms of Epo may have an important role in therapy.

The erythropoietin-sensitive membrane phosphoprotein, pp43, is a protein serine/threonine kinase.

We have shown previously that treatment of isolated erythroid cell plasma membranes with erythropoietin leads to a rapid decrease in pp43, an erythropoietinsensitive membrane phosphoprotein (Choi, H. S., Wojchowski, D. M., and Sytkowski, A. J., J. Biol. Chem. 262, 2933, 1987; Choi, H. S., Bailey, S. C., Donahue, K. A., Vanasse, G. J., and Sytkowski, A. J., J. Biol. Chem. 265, 4143, 1990). We have now demonstrated this effect in intact cells and have obtained further information regarding pp43 function during erythropoietin stimulation. 32P-phosphorylated membranes were subjected to conditions of increasing pH. [32P]pp43 dissociated readily into solution, reaching half-maximal dissociation at pH approximately 9. This dissociation was enhanced markedly by increasing the ionic strength up to a maximum of 0.5 M KCl. These biochemical properties characterize pp43 as a membrane-associated protein. Addition of [gamma-32P]ATP to an aqueous supernatant prepared from unlabeled membranes resulted in the 32P-phosphorylation of pp43 in solution, after dissociation from the plasma membrane. Furthermore, erythropoietin treatment of unlabeled, intact cells followed by fractionation and 32P-phosphorylation resulted in a striking erythropoietin- and time-dependent increase in [32P]pp43 found in the supernatant and a concomitant decrease in [32P]pp43 found in the membrane pellet. This strongly suggests that erythropoietin stimulates the dissociation of pp43 from the plasma membrane and promotes translocation into the supernatant (cytoplasm). Using a renaturation kinase assay, we demonstrated that pp43 is capable of autophosphorylation on serine and threonine, thus identifying it as a new protein serine/threonine kinase. The results suggest a role for pp43 in transmembrane signaling.

Protein kinase C-epsilon is necessary for erythropoietin's up-regulation of c-myc and for factor-dependent DNA synthesis. Evidence for discrete signals for growth and differentiation.

Erythropoietin regulates the transcription of the protooncogenes c-myc and c-myb by discrete protein kinase C (PKC)-dependent and protein serine/threonine phosphatase-dependent pathways, respectively (Spangler, R., Bailey, S. C., and Sytkowski, A. J. (1991) J. Biol. Chem. 266, 681-684; Patel H. R, Choi H.-S, and Sytkowski A. J. (1992) J. Biol. Chem. 267, 21300-21302). In the present study we demonstrate that up-regulation of c-myc requires the PKC-epsilon isoform and that this pathway is required for erythropoietin-induced DNA synthesis (growth) but apparently not for beta-globin expression (differentiation). Treatment of Rauscher murine erythroleukemia cells resulted in phosphorylation of phospholipase C-gamma1 and activation of PKC-epsilon as evidenced by its translocation from soluble to particulate subcellular fractions. Artificial down-regulation of PKC-epsilon with antisense oligodeoxynucleotides blocked erythropoietin's up-regulation of c-myc in a concentration-dependent manner. In contrast, antisense oligodeoxynucleotides to PKC-alpha, -beta, -gamma, -delta, and -zeta had no effect. Although down-regulation of PKC-epsilon blocked the increase in c-myc expression, it did not inhibit erythropoietin induction of beta-globin expression, a marker of erythroid differentiation. However, down-regulation of PKC-epsilon did block factor-dependent DNA synthesis quantified by measurement of [3H]thymidine incorporation into newly synthesized DNA of normal murine erythroid cells. The results are consistent with discrete intracellular signals regulating erythroid cell growth and differentiation.

Functional and structural role of arginine 103 in human erythropoietin.

The identification of amino acid residues essential for function of the hematopoietic growth factor erythropoietin has been approached by several methods, including comparisons of related sequences, immunochemical approaches, mutagenesis, and computer modeling. We have reported previously that mutations within amino acids 100-109 of erythropoietin can have profound effects on the hormone's structure and/or activity and that Arg103 is especially important for function [Y. Chern, T. Chung, and A. J. Sytkowski (1991) Eur. J. Biochem. 202, 225-229; J. Grodberg, K.L. Davis, and A. J. Sytkowski (1993) Eur. J. Biochem. 218, 597-601]. We have now constructed a series of Arg103 substitutions in order to determine the structural features of amino acid 103 required for biological activity. Each of the mutants was expressed and secreted efficiently by transfected COS1 cells. Mutants Arg103Asn, Arg103Gln, and Arg103Glu exhibited no biological activity. In contrast, Arg103His and Arg103Lys had specific activities equal to 2 and 25%, respectively, of that of wild-type erythropoietin, indicating that a positive charge may be required at position 103 but that other constraints necessitate the presence of Arg for full activity. A role for amino acid 103 in the protein's structure was supported by the results of experiments which revealed marked differences in heat stability among the mutants. We hypothesize that an Arg at position 103 may confer sufficient flexibility to the receptor binding domain to facilitate initial binding to the receptor and may then stabilize the binary complex by hydrogen bonding with carbonyls of the receptor protein.

Cloning differentially expressed genes by linker capture subtraction.

We have developed a simple and effective method, designated linker capture subtraction (LCS), for cloning differentially expressed genes between two cell types or between cells treated in two different ways. In the first step of the method, two mRNA populations are converted to double-stranded cDNAs, fragmented, and ligated to linkers for PCR amplification. In the second step, the linkered DNA (tester) from one mRNA population is hybridized to an excess of the unlinkered DNA (driver) from the other mRNA population, followed by incubation with mung bean nuclease which digests single-stranded DNA specifically. This leaves only tester-tester homohybrids to be amplified by PCR in the following step, so as to achieve an enrichment of tester-specific sequences. The amplified PCR products are then used as tester for another round of subtraction. The process of subtraction is carried out three times, and the final PCR products are inserted into a vector for clonal analysis. We have used the strategy to begin to clone and identify the genes expressed differentially between the human prostate cancer cell lines LNCaP and PC-3, which have different tumorigenic and metastatic potentials. We demonstrated strong enrichment of target sequences. We also report the identities of two of the genes expressed differentially in these cell lines. One is prostate-specific antigen (PSA) which is known to be expressed in LNCaP but not in PC-3. The other is vimentin, the differential expression of which has not been reported previously in these prostate cancer cells.

Erythropoietin activation of AP1 (Fos/Jun).

Erythropoietin (Epo) is the principal natural inducer of erythroid differentiation. The mechanisms by which signals generated at the Epo receptor (Epo-R) are transmitted to the nucleus are being explored. We now report that Epo strongly increases the activity of the transcription factor AP1 in both transformed and normal erythroid cells. Using antibodies to Fos and Jun, we have found that the Epo-induced AP1 heterodimer is composed primarily of authentic Fos and Jun proteins. Blocking protein kinase C (PKC) activity with H7 completely prevented the increase in AP1 activity in response to Epo. Importantly, the increase in AP1 activity was not due to increased expression of either c-fos or c-jun, as evidenced by the steady-state mRNA levels of both genes. Our results suggest that Epo may induce AP1 activity via a co- or posttranslational mechanism, presumably through modification of the Fos and/or Jun proteins.

Regulated production of a pleiotropic cytokine-platelet-derived growth factor--by differentiating erythroid cells in vitro and in vivo.

Erythroid progenitor growth in vitro is stimulated by exogenous platelet-derived growth factor (PDGF). We now report that both normal and transformed erythroid progenitor cells produce authentic PDGF in vitro and in vivo. Importantly, this production is highly regulated during erythropoiesis. Addition of soluble lysates from Rauscher murine erythroleukemia cells--an erythropoietin-responsive model progenitor cell line--to quiescent BALB/c 3T3 fibroblasts resulted in a mitogenic response identical to that observed with the addition of authentic recombinant PDGF. Polyclonal and monoclonal anti-PDGF antibodies immunoabsorbed 50-100% of this activity. Induction of Rauscher cell differentiation in vitro with dimethyl sulfoxide or erythropoietin for 48-72 hr markedly upregulated PDGF production by 17- to 18-fold and 14- to 38-fold, respectively. Importantly, stimulation of normal erythropoiesis in vivo in mice treated either with phenylhydrazine or with erythropoietin increased PDGF levels in the spleen by 11- to 48-fold and 20- to 34-fold, respectively. These results strongly suggest a role for erythroid cell-derived PDGF in normal erythropoiesis and provide documentation of the regulated production of a pleiotropic cytokine by erythroid cells.

Constitutive secretion of erythropoietin by human renal adenocarcinoma cells in vivo and in vitro.

The paraneoplastic syndrome of erythrocytosis is associated with a variety of neoplasms including renal adenocarcinoma, cerebellar hemangioma, and hepatoma. We now report the characterization of the biological and molecular features of an erythropoietin-secreting human renal adenocarcinoma, designated RCCEp+. Serial transplantation of the tumor in athymic mice resulted in a dramatic increase in hematocrit and serum erythropoietin concentration. Growth in vitro was accompanied by a constant rate of erythropoietin secretion. Karyotype analysis demonstrated several unusual features, including the absence of 3p deletions and near tetraploidy. Erythropoietin mRNA was demonstrated by Northern blot both in freshly excised tumor and in tumor cells growing in vitro. Erythropoietin secretion was constitutive and was not induced either by cobalt or hypoxia. Southern blot analysis revealed no rearrangement of the erythropoietin gene in the tumor. Interestingly, in situ hybridization demonstrated erythropoietin mRNA in only a small population of the tumor cells. Further studies of RCCEp+ should prove useful in elucidating the molecular basis for this paraneoplastic syndrome.