A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells.

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.

Quantification of the spatiotemporal microstructural organization of the human brain association, projection and commissural pathways across the lifespan using diffusion tensor tractography.

Using diffusion tensor tractography, we quantified the microstructural changes in the association, projection, and commissural compact white matter pathways of the human brain over the lifespan in a cohort of healthy right-handed children and adults aged 6-68 years. In both males and females, the diffusion tensor radial diffusivity of the bilateral arcuate fasciculus, inferior longitudinal fasciculus, inferior fronto-occipital fasciculus, uncinate fasciculus, corticospinal, somatosensory tracts, and the corpus callosum followed a U-curve with advancing age; fractional anisotropy in the same pathways followed an inverted U-curve. Our study provides useful baseline data for the interpretation of data collected from patients.

Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells?

Angiomyolipomas are benign tumors of the kidney which express phenotypes of smooth muscle, fat, and melanocytes. These tumors appear with increased frequency in the autosomal dominant disorder tuberous sclerosis and are the leading cause of morbidity in adults with tuberous sclerosis. While benign, these tumors are capable of provoking life threatening hemorrhage and replacement of the kidney parenchyma, resulting in renal failure. The histogenesis of these tumors is currently unclear, although currently, we believe these tumors arise from "perivascular epithelioid cells" of which no normal counterpart has been convincingly demonstrated. Recently, stem cell precursors have been recognized that can give rise to smooth muscle and melanocytes. These precursors have been shown to express the neural stem cell marker NG2 and L1. In order to determine whether angiomyolipomas, which exhibit smooth muscle and melanocytic phenotypes, express NG2 and L1, we performed immunocytochemistry on a cell line derived from a human angiomyolipoma, and found that these cells are uniformly positive. Immunohistochemistry of human angiomyolipoma specimens revealed uniform staining of tumor cells, while renal cell carcinomas revealed positivity only of angiogenic vessels. These results support a novel histogenesis of angiomyolipoma as a defect in differentiation of stem cell precursors.

Nuclear localization of HBD-1 in human keratinocytes.

OBJECTIVE: Human defensins and cathelicidins are a family of cationic antimicrobial peptides (AMPs), which play multiple roles in both innate and adaptive immune systems. They have direct antimicrobial activity against several microorganisms including burn pathogens. The majority of components of innate and adaptive immunity either express naturally occurring defensins or are otherwise chemoattracted or functionally affected by them. They also enhance adaptive immunity and wound healing and alter antibody production. All mechanisms to explain multiple functions of AMPs are not clearly understood. Prior studies to localize defensins in normal and burned skin using deconvolution fluorescence scanning microscopy indicate localization of defensins in the nucleus, perinuclear regions, and cytoplasm. The objective of this study is to further confirm the identification of HBD-1 in the nucleus by deconvolution microscopic studies involving image reconstruction and wire frame modeling. RESULTS: Our study demonstrated the presence of intranuclear HBD-1 in keratinocytes throughout the stratum spinosum by costaining with the nuclear probe DAPI. In addition, HBD-1 sequence does show some homology with known cationic nuclear localization signal sequences. CONCLUSION: To our knowledge, this is the first report to localize HBD-1 in the nuclear region, suggesting a role for this peptide in gene expression and providing new data that may help determine mechanisms of defensin functions.

Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation.

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3' poly(A) tail-PABP complex.

THE MECHANISM OF TUMORIGENESIS IN THE IMMORTALIZED HUMAN PANCREATIC CELL LINES: CELL CULTURE MODELS OF HUMAN PANCREATIC CANCER

The mechanism of tumorigenesis in the immortalized human pancreatic cell lines: cell culture models of human pancreatic cancer Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer in the world. The most common genetic lesions identified in PDAC include activation of K-ras (90%) and Her2 (70%), loss of p16 (95%) and p14 (40%), inactivation p53 (50-75%) and Smad4 (55%). However, the role of these signature gene alterations in PDAC is still not well understood, especially, how these genetic lesions individually or in combination contribute mechanistically to human pancreatic oncogenesis is still elusive. Moreover, a cell culture transformation model with sequential accumulation of signature genetic alterations in human pancreatic ductal cells that resembles the multiple-step human pancreatic carcinogenesis is still not established. In the present study, through the stepwise introduction of the signature genetic alterations in PDAC into the HPV16-E6E7 immortalized human pancreatic duct epithelial (HPDE) cell line and the hTERT immortalized human pancreatic ductal HPNE cell line, we developed the novel experimental cell culture transformation models with the most frequent gene alterations in PDAC and further dissected the molecular mechanism of transformation. We demonstrated that the combination of activation of K-ras and Her2, inactivation of p16/p14 and Smad4, or K-ras mutation plus p16 inactivation, was sufficient for the tumorigenic transformation of HPDE or HPNE cells respectively. We found that these transformed cells exhibited enhanced cell proliferation, anchorage-independent growth in soft agar, and grew tumors with PDAC histopathological features in orthotopic mouse model. Molecular analysis showed that the activation of K-ras and Her2 downstream effector pathways –MAPK, RalA, FAK, together with upregulation of cyclins and c-myc were involved in the malignant transformation. We discovered that MDM2, BMP7 and Bmi-1 were overexpressed in the tumorigenic HPDE cells, and that Smad4 played important roles in regulation of BMP7 and Bmi-1 gene expression and the tumorigenic transformation of HPDE cells. IPA signaling pathway analysis of microarray data revealed that abnormal signaling pathways are involved in transformation. This study is the first complete transformation model of human pancreatic ductal cells with the most common gene alterations in PDAC. Altogether, these novel transformation models more closely recapitulate the human pancreatic carcinogenesis from the cell origin, gene lesion, and activation of specific signaling pathway and histopathological features.

EXPRESSION AND IMMUNOLOGICAL CHARACTERIZATION OF HERV-K TRANSMEMBRANE ENVELOPE PROTEIN IN HUMAN BREAST CANCER

The human endogenous retrovirus K (HERV-K) env gene encodes envelope protein comprising surface (SU) and transmembrane (TM) domains. Having shown the exclusive expression of SU in human breast cancer and the stimulation of SU-specific immune responses in patients with breast cancer, our research here confirmed and extended the data by investigating the expression of HERV-K TM envelope domain and the induction of specific immune responses against TM in breast cancer patients. We found HERV-K TM mRNA and protein expression only in human breast cancer cells but not in normal controls. The specific immune responses against TM domain were induced in mice determined by enzyme-linked immunosorbent assay (ELISA) and IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay. Furthermore, ELISA detected higher titers of anti-HERV-K TM Env IgG antibodies in sera of breast cancer patients. In addition, the magnitude of the anti-HERV TM B cell response was correlated with the disease stage. Peripheral blood mononuclear cells (PBMCs) before and after in vitro stimulation (IVS) with HERV-K TM from patients with breast cancer as well as healthy controls were tested for T cell responses against HERV-K TM domain by ELISPOT assay. Breast cancer patients (n=21) had stronger HERV-K TM-specific cellular responses than healthy controls (n=12) (P < 0.05). These findings suggest, for the first time, that HERV-K TM expression was enhanced in human breast cancer cells and was able to induce specific B cell and T cell immune responses in breast cancer patients. This study provides support for HERV-K TM as a promising source of antigen for anti-tumor immunotherapy, prevention, diagnosis, and prognosis.

Differentiative requirements for human CD8$\sp+$ suppressor lymphocytes

The aim of this research was to characterize the differentiative requirements of human CD8$\sp{+}$ suppressor lymphocytes. We investigated the role of monocytes in cellular interactions required for generation of T suppressor cells (Ts) in pokeweed mitogen (PWM) stimulated peripheral blood mononuclear cells (PBMC). We observed that the functional activity of CD8$\sp{+}$ T cells was dependent on the concentration of monocytes in the inductive cultures; at concentrations normally present in peripheral blood, PWM stimulation induced potent suppressor activity, whereas under conditions of moderate monocyte depletion the same phenotypic subset of CD8$\sp{+}$ cells enhanced responses. We also demonstrated that differentiation of CD8$\sp{+}$CD28$\sp{-}$ suppressor cells could be mediated by soluble products elaborated by monocytes and CD4$\sp{+}$ cells, identified as PGE$\sb2$ and IFN$\gamma$ respectively. These two signals were required sequentially to cause Ts induction. That is PGE$\sb2$ was required initially, followed by an IFN$\gamma$-dependent differentiative step. We also explored the possibility that PGE$\sb2$ caused modulation of the IFN$\gamma$ receptor number and/or affinity on CD8$\sp{+}$ cells, which might render these cells responsive to the differentiative effect of the IFN$\gamma$-signal. Using radiolabelled $\sp{125}$I-IFN$\gamma$, direct binding assays demonstrated that 10$\sp{-8}$M PGE$\sb2$ selectively increased the number of receptors on the CD8$\sp{+}$ cells. In contrast CD4$\sp{+}$ cells treated similarly exhibited no significant change in their number of IFN$\gamma$ receptors. These results, thus, suggest a relationship between PGE$\sb2$ induced expression of IFN$\gamma$ receptor and the initial requirement for PGE$\sb2$ in IFN$\gamma$-dependent differentiation of Ts cells. Together, our results suggest a crucial role for PGE$\sb2$ and IFN$\gamma$ in regulation of the immune response. Furthermore, such detailed definition of the differentiative requirements for CD8$\sp{+}$ suppressor cells should provide new insight into fundamental mechanisms of immunoregulation. ^

Stability of specific immunoglobulin secretion by EBV-transformed lymphoblastoid cells and human-murine heterohybridomas

The purpose of this project was to determine if stability of specific antibody secretion improved after fusion of Epstein-Barr virus (EBV)-transformed lymphoblastoid cells with P3X63Ag8.653 murine myeloma cells. Production of human monoclonal antibodies by Epstein-Barr virus transformation and somatic cell fusion has been used by many laboratories, however the steps involved have not been fully optimized. B lymphocytes isolated from the peripheral blood of normal donors were enriched for Thomsen-Friedenreich (T) antigen-reactive cells by panning on asialoglycophorin. The EBV-transformed lymphoblastoid cell lines generated from asialoglycophorin-adherent B lymphocytes were treated in three different manners: (1) cloned and maintained in culture as monoclonal lymphoblastoid cell lines, (2) cloned and fused with murine myeloma cells or (3) fused shortly after transfomation without prior cloning. Cloned lymphoblastoid cell lines maintained in culture without fusion either died or lost specific antibody secretion within five months. Uncloned lymphoblastoid cells remained viable for up to three months but lost specific antibody secretion within two months probably due to overgrowth by nonspecific clones. In an attempt to increase longevity and to stabilize specific antibody secretion by these cells, the cloned lymphoblastoid cells were fused with murine myeloma cells. In nine of ten fusions no hybrids were recovered. As an alternate approach, uncloned lymphoblastoid cells secreting T antigen-specific antibody were hybridized with murine myeloma cells, hybrids secreting T antigen-specific antibody were recovered in six of seven fusions. Furthermore, T antigen-specific antibodies of high titer were secreted by the heterohybridoma clones for more than five months of continuous culture. These heterohybridoma cells secreted more immunoglobulin, produced greater titers of antibody and maintained specific antibody secretion longer than either monoclonal or polyclonal EBV-transformed lymphoblastoid cells. These studies have conclusively demonstrated that fusion of polyclonal lymphoblastoid cells secreting T antigen-specific antibody with murine myeloma cells results in prolongation of human monoclonal antibody production compared with unfused monoclonal or polyclonal lymphoblastoid cell lines. This procedure should be generally applicable for the production of stable human monoclonal antibody-secreting cells lines from peripheral blood lymphocytes. ^

Characterization and mechanism of action of suppressor factors derived from human T cell hybridomas

This laboratory developed human T-cell hybridomas which constitutively secrete suppressor factors (SF) capable of inhibiting immune responses (Hybridoma 6:589 (1987). The mechanisms by which human T-cell hybridoma-derived SFs (designated 160 and 169) and Jurkat leukemic T-cell line derived SF inhibit the proliferative response to mitogen by human PBMC were investigated. The Jurkat SF had a pI of 5.2 whereas the 160 and 169 SF had pI of 5.7 and 4.7 (two peaks) and 4.7, respectively. The SF was not transforming growth factor-beta based upon neutralization and iummunoprecipitation experiments with anti-TGF-beta polyclonal antibody. Il-2 production by human PBMC cultured with Con A or OKT3 mAb in the presence of SF was found to be inhibited by greater than 80%. The proliferative responses of SF treated PBMC could not be restored by addition of exogeneous human IL-2. Inhibition of the proliferative responses could not be reversed by addition of exogenous rIL-1, rIL-2 or rIL-4 alone or in paired combinations. The expression of IL-2 receptors (TAC Ag) on Con A activated cultures time points was not affected by treatment with any SFs. Both the 160 and 169 hybridoma-derived SFs were found to arrest PHA induced cell cycle progression in G$\sb0$/G$\sb1$ phase, whereas SF from the Jurkat T-cell line arrested progression in the S phase. Pretreatment of PBMC with SF prior to the addition of mitogen, followed by washing, did not alter the proliferative response of these PBMC nor their cell cycle progression suggesting that cell activation is necessary for these SF to inhibit proliferative responses. Northern blot analysis of total mRNA from mitogen stimulated PBMC in the presence of SF, revealed a time dependent accumulation of an IL-2 specific mRNA of increased size (2.8 kB) in addition to the expected 1.0 kB mature IL-2 message. Interferon-gamma mRNA was of the appropriate size but its half-life was prolonged in SF treated cultures. IL-2 receptor and IL-1 beta mRNA expression was not altered in these cells. ^

The molecular cloning and characterization of human heparanase cDNA and the immunochemical localization of heparanase in metastatic melanomas

Heparanase, an endo-$\beta$-D-glucuronidase, has been associated with melanoma metastasis. Polyclonal antibodies directed against the murine N-terminal heparanase peptide detected a M$\sb{\rm r}\sim 97,000$ protein upon SDS-polyacrylamide gel electrophoresis of mouse melanoma and human melanoma cell lysates. In an indirect immunocytochemical study, metastatic human A375-SM and mouse B16-BL6 melanoma cells were stained with the anti-heparanase antibodies. Heparanase antigen was localized in the cytoplasm of permeabilized melanoma cells as well as at the cell surface of unpermeabilized cells. Immunohistochemical staining of frozen sections from syngeneic mouse organs containing micrometastases of B16-BL6 melanoma demonstrated heparanase localized in metastatic melanoma cells, but not in adjacent normal tissues. Similar studies using frozen sections of malignant melanomas resected from patients indicated that heparanase is localized in invading melanoma cells, but not in adjacent connective tissues.^ Monoclonal antibodies directed against murine heparanase were developed and characterized. Monoclonal antibody 10E5, an IgM, precipitated and inhibitated the enzymatic activity of heparanase. A 2.6 kb cDNA was isolated from a human melanoma $\lambda$gt11 cDNA library using the monoclonal antibody 10E5. Heparan sulfate cleavage activity was detected in the lysogen lysates from E. Coli Y1089 infected with the $\lambda$gt11 cDNA and this activity was inhibited in the presence of 10-fold excess of heparin, a potent inhibitor of heparanase. The nucleotide sequence of the cDNA was determined and insignificant homology was found with the gene sequences currently known. The cDNA hybridized to a 3.2-3.4 kb mRNA in human A375 melanoma, WI-38 fibroblast, and THP-1 leukemia cells using Northern blots.^ Heparanase expression was examined using Western and Northern blots. In comparison to human A375-P melanoma cells, the quantity of 97,000 protein recognized by the polyclonal anti-heparanase antibodies doubled in the metastatic variant A375-SM cells and the quantity of 3.2-3.4 kb mRNA doubled in A375MetMix, a metastatic variant similar to A375-SM cells. In B16 murine melanoma cell, the intensity of the 97,000 protein increased more than 2 times comparing with B16-F1 cells. The extent in the increase of the protein and the mRNA levels is comparable to the change of heparanase activity observed in those cells.^ In summary, the studies suggest that (a) the N-terminus of the heparanase molecule in mouse and human is antigenically related; (b) heparanase antigens are localized at the cell surface and in the cytoplasm of metastatic human and mouse melanoma cells; (c) heparanase antigens are localized in invasive and metastatic murine and human melanomas in vivo, but not in adjacent normal tissues; (d) heparanase molecule appeared to be differentially expressed at the transcriptional as well as at the translational level; and (e) the size of human heparanase mRNA is 3.2-3.4 kilobase. ^

Antisense-mediated inhibition of papillomavirus type-18 in human squamous cell carcinoma

Numerous co-factors, genetic, environmental and physical, play an important role in development and prognosis of cancer. Each year in the USA, more than 31,000 cases of oral and 13,000 cases of cervical cancer are diagnosed. Substantial epidemiological data supports a high correlation between development of these cancers and the presence of specific types of human papillomaviruses (HPV). Molecular biological studies show that not only are several of the viral genes necessary and sufficient to cause transformation but they also function synergistically with other co-factors. Evidence suggests that prevention of infection or inhibition of viral gene expression may alter the course of malignant transition. The main objective of this project was to test the hypothesis that some human carcinoma cells, containing HPV, behave in malignant manner because the viral genes function in the maintenance of some aspect of the transformed phenotype.^ The specific aims were (1) to select oral and cervical cancer cell lines which were HPV-negative or which harbored transcriptionally active HPV-18, (2) to construct and determine the effects of recombinant sense or antisense expressing vectors, (3) to test the effects of synthetic antisense oligodeoxynucleotides on the transformed behavior of these cells.^ To screen cells, we performed Southern and Northern analysis and polymerase chain reactions. When antisense-expressing vectors were used, cells harboring low numbers of HPV-18 where unable to survive transfection but they were readily transfected with all other constructs. Rare antisense transfectants obtained from HPV-positive cells showed significantly altered characteristics including malignant potential in nude mice. The HPV-negative cells showed no differences in transfection efficiencies or growth characteristics with any construct.^ In addition, treatment of the HPV-positive cells with antisense, but not random oligodeoxynucleotides, resulted in decreased cell proliferation and even cell death. These effects were dose-dependent, synergistic and HPV-specific.^ These results suggest that expression of viral genes play an important role in the maintenance of the transformed phenotype which implies that inhibition of expression, by antisense molecules, may be therapeutic in HPV-induced tumors. ^

Nuclear proteins interacting with an AP-1/CRE-like promoter sequence in the human TNF$\alpha$ gene

Monocyte developmental heterogeneity is reflected at the cellular level by differential activation competence, at the molecular level by differential regulation of gene expression. LPS activates monocytes to produce tumor necrosis factor-$\alpha$ (TNF). Events occurring at the molecular level necessary for TNF regulation have not been elucidated, but depend both on activation signals and the maturation state of the cell: Peripheral blood monocytes produce TNF upon LPS stimulation, but only within the first 72 hours of culture. Expression of c-fos is associated with monocytic differentiation and activation; the fos-associated protein, c-jun, is also expressed during monocyte activation. Increased cAMP levels are associated with down regulation of macrophage function, including LPS-induced TNF transcription. Due to these associations, we studied a region of the TNF promoter which resembles the binding sites for both AP-1(fos/jun) and CRE-binding protein (or ATF) in order to identify potential molecular markers defining activation competent populations of monocytic cells.^ Nuclear protein binding studies using extracts from THP-1 monocytic cells stimulated with LPS, which stimulates, or dexamethasone (Dex) or pentoxyfilline (PTX), which inhibit TNF production, respectively, suggest that a low mobility doublet complex may be involved in regulation through this promoter region. PTX or Dex increase binding of these complexes equivalently over untreated cells; approximately two hours after LPS induction, the upper complex is undetectable. The upper complex is composed of ATF2 (CRE-BP1); the lower is a heterodimer of jun/ATF2. LPS induces c-jun and thus may enhance formation of jun-ATF2 complexes. The simultaneous presence of both complexes may reduce the amount of TNF transcription through competitive binding, while a loss of the upper (ATF2) and/or gain of the lower (jun-ATF2) allow increased transcription. AP-1 elements generally transduce signals involving PKC; the CRE mediates a cAMP response, involving PKA. Thus, this element has the potential of receiving signals through divergent signalling pathways. Our findings also suggest that cAMP-induced inhibition of macrophage functions may occur via down regulation of activation-associated genes through competitive binding of particular cAMP-responsive nuclear protein complexes. ^

The processing of human pro-tumor necrosis factor

Human pro-TNF-$\alpha$ is a 26 kd type II transmembrane protein, and it is the precursor of 17 kd mature TNF. Pro-TNF release mature from its extracellular domain by proteolytic cleavage between resideu Ava ($-$1) and Val (+1). Both forms of TNF are biologically active and the native form of mature TNF is a bell-shaped trimer. The structure of pro-TNF was studied both in intact cell system and in an in vitro translation system by chemical crosslinking. We found that human pro-TNF protein exist as a trimer in intact cells (LPS-induced THP-1 cells and TNF cDNA transfected COS-3 cells) and this trimeric structure is assembled intracellularly, possibly in the ER. By analysis several deletion mutants, we observed a correlation between expression of pro-TNF cytotoxicity in a juxtacrine fashion and detection of the trimer, suggesting the trimeric structure is very important for its biologic activity. With a series of deletion mutants in the linking domain, we found that the small deletion did not block the cleavage and large deletion did regardless of the presence or absence of the native cleavage site, suggesting that the length of the residues between the plasma membrane and the base of the trimer determines the rate of the cleavage, possibly by blocking the accessibility of the cleavage enzyme to its action site. Our data also suggest that the native cleavage site is not sufficient for the release of mature TNF and alternative cleavage site(s) exists. ^

Involvement of heparin-like glycosaminoglycans and expression of a novel heparan sulfate binding protein (p24) during human placentation and in a model for human implantation

Previous studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. To investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were selected to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay showed that initial binding between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAG) with heparin-like properties, i.e., heparan sulfate and dermatan sulfate. Furthermore, a single class of highly specific, protease-sensitive heparin/heparan sulfate binding sites exist on the surface of RL95 cells. Three heparin binding, tryptic peptide fragments were isolated from RL95 cell surfaces and their amino termini partially sequenced. Reverse transcription-polymerase chain reaction (RT-PCR) generated 1 to 4 PCR products per tryptic peptide. Northern blot analysis of RNA from RL95 cells using one of these RT-PCR products identified a 1.2 Kb mRNA species (p24). The amino acid sequence predicted from the cDNA sequence contains a putative heparin-binding domain. A synthetic peptide representing this putative heparin binding domain was used to generate a rabbit polyclonal antibody (anti-p24). Indirect immunofluorescence studies on RL95 and JAR cells as well as binding studies of anti-p24 to intact RL95 cells demonstrate that p24 is distributed on the cell surface. Western blots of RL95 membrane preparations identify a 24 kDa protein (p24) highly enriched in the 100,000 g pellet plasma membrane-enriched fraction. p24 eluted from membranes with 0.8 M NaCl, but not 0.6 M NaCl, suggesting that it is a peripheral membrane component. Solubilized p24 binds heparin by heparin affinity chromatography and $\sp{125}$I-heparin binding assays. Furthermore, indirect immunofluorescence studies indicate that cytotrophoblast of floating and attached villi of the human fetal-maternal interface are recognized by anti-p24. The study also indicates that the HSPG, perlecan, accumulates where chorionic villi are attached to uterine stroma and where p24-expressing cytotrophoblast penetrate the stroma. Collectively, these data indicate that p24 is a cell surface membrane-associated heparin/heparan sulfate binding protein found in cytotrophoblast, but not many other cell types of the fetal-maternal interface. Furthermore, p24 colocalizes with HSPGs in regions of cytotrophoblast invasion. These observations are consistent with a role for HSPGs and HSPG binding proteins in human trophoblast-uterine cell interactions. ^