Downregulation Of CD44 Research Articles

Enhanced GM3 expression, associated with decreased invasiveness, is induced by brefeldin A in bladder cancer cells

We reported previously that non-invasive bladder cancer expresses high level of GM3 ganglioside, whereas invasive tumors have low levels. Since glycosphingolipid synthesis in Golgi is modified greatly by a macrocyclic lactone isolated from fungi, brefeldin A (BFA), we studied effects of BFA on expression of glycosphingolipids and on invasiveness of bladder cancer cell lines. Only GM3 synthesis in invasive tumors was greatly enhanced upon treatment with BFA; synthesis of other glycosphingolipids with lacto-series type 2 or globo-series structure in both invasive and non-invasive tumors was not changed. Invasiveness of bladder cancer cells was greatly decreased in association with the great increase of GM3 synthesis induced by BFA treatment. Level of sialyl-Lex expressed in invasive cell line YTS1, which provides the adhesive property of the cells to E-selectin, was unchanged upon BFA treatment. All the bladder cancer cell lines, regardless of invasiveness, highly express tetraspanin CD9. GM3 has been implicated as a co-factor of CD9 in control of tumor cell motility. Down-regulation of CD9 is associated with metastatic properties of tumor cells and survival of patients with colonic cancer. Therefore, enhanced synthesis of GM3 induced by BFA, causing decrease of invasiveness in bladder cancer, is ascribable to the capability of GM3 to interconnect integrin with CD9, in analogy to colonic cancer and perhaps many other types of cancer.

Hypermethylation of the CD44 gene is associated with progression and metastasis of human prostate cancer.

CD44 is a metastasis suppressor gene for prostate cancer and the down-regulation of CD44 and its variants is associated with the progression of prostate cancer. Also, hypermethylation of the CpG islands of the CD44 gene is closely associated with transcriptional inactivation, resulting in the decreased expression of CD44. To clarify the exact role of methylation status of CpG islands of CD44 gene in the progression and metastasis of prostate cancer, we investigated the methylation status of this gene in primary and metastatic human prostate tumors obtained from surgery or autopsy. We examined 97 samples from 40 Japanese patients with adenocarcinoma of the prostate. Tumor tissues were obtained from radical prostatectomy specimens from eight patients with stage B, 12 patients with stage C and three patients with stage D1 and at autopsy from 17 hormone-refractory metastatic cases, who had initially responded to the therapy and thereafter relapsed. Distant metastatic tissues were also obtained at autopsy (i.e., liver, lung, kidney, mammary gland, and pelvic lymph nodes) from 10 of 17 hormone-refractory cases. We analyzed the hypermethylation status of CD44 promotor region by PCR using genomic DNAs digested with the m(5)C-sensitive restriction enzyme HpaII. The correlation between the methylation status of CD44 gene and the stage progression of prostate cancer was statistically significant (P = 0.0438). In two of 10 hormone-refractory cases, a comparison of the methylation status of the CD44 gene in metastases to that in primary tumors revealed interfocal heterogeneity of CD44 methylation status. These results indicate an important role of CD44 methylation in the progression and metastasis of prostate cancer, although the amount of methylational heterogeneity is substantial among metastatic sites within the same patient.

Expression of CD44 and its isoforms in the fetal neuroblasts.

CD44 is a polymorphic transmembrane glycoprotein that exists as multiple isoforms resulting from alternative splicing and posttranslational modifications. Enhanced expression of CD44 has been correlated to the tumorigenicity and metastatic behavior in different malignant tumors. In contrast, human neuroblastomas exhibit an inverse correlation between CD44 expression and tumor malignancy. To determine whether there is a CD44 silencing in sympatho-adrenal precursors as a possible explanation for the down-regulation of CD44 in neuroblastomas, the expression of standard CD44H and v6, v7, v7v8, or v10 isoforms was analyzed by immunohistochemistry in human adrenal glands of 14- to 20-week-old gestational age fetuses. All of the fetal neuroblasts localized in the adrenal gland parenchyma and migrating from the sympathetic nerve chain into the fetal adrenal cortex strongly expressed CD44H but none of the CD44 isoforms could be detected in these cells. In contrast, a strong expression of CD44v7 and v6 was detected in the fetal adrenal cells. These results indicate that, as for many other cell types, the CD44H expressed by fetal neuroblasts may contribute to controlling their migration into the adrenal medulla and that the down-regulation of CD44H in neuroblastoma cells should be explained by mechanisms other than the fetal suppression of CD44H expression in their normal counterparts.

Reduced CD43 Expression on the Neutrophils of MDS Patients Correlates With an Activated Phenotype of These Cells

CD43 (also known as leukosialin and sialophorin) is a surface sialoglycoprotein expressed at high levels on most leukocytes implicated in adhesion, antiadhesion, and activation/proliferation mechanisms. We studied the expression of this molecule on the leukocytes of patients with myelodysplastic syndromes (MDSs) in an effort to detect acquired deficiencies of this molecule. We used immunofluorescence flow cytometry in analyzing whole blood and isolated neutrophils from 49 MDS patients, 33 men and 16 women aged 33 to 85 years (median, 75 years), and 18 healthy individuals aged 35 to 80 years (median, 72 years). According to French-American-British classification criteria, 13 patients had refractory anemia, 18 had refractory anemia with ringed sideroblasts, 9 had refractory anemia with excess of blasts, 4 had refractory anemia with excess of blasts in transformation to acute leukemia, and 5 had chronic myelomonocytic leukemia. We found decreased expression of CD43 on the neutrophils of these patients, and we correlated this finding with the activation status of these cells as it is defined by their phenotypes. We studied the expression of CD11b, CD18, CD35, CD67, CD69, CD44, and CD53 molecules known to be changed in the activated form of neutrophils. CD43 expression correlated positively with CD53 and CD44 expression and negatively with CD11b, CD18, CD35, CD67, and CD69 expression. Additionally, increased levels of soluble vascular cell adhesion molecules were detected in these patients, suggesting endothelial cell activation. In conclusion, we believe that the decreased expression of CD43 on the neutrophils of MDS patients is associated with activation of these cells and is probably due to cleavage of the molecule from the cell surface and that the same mechanism is possibly responsible for the parallel down-regulation of CD44 and CD53.

The BRG-1 Subunit of the SWI/SNF Complex Regulates CD44 Expression

Aberrant regulation of CD44, a transmembrane glycoprotein, has been implicated in the growth and metastasis of numerous tumors. Although both CD44 overexpression and loss have been implicated in tumor progression, the mechanism of CD44 down-regulation in these tumor types is not known. By immunoblot and reverse transcription-polymerase chain reaction analysis we determined that a cervical carcinoma cell line, C33A, lacks CD44 expression. To determine how CD44 is down-regulated in C33A cells, we utilized cell fusions of C33A cells with a CD44-expressing cell line (SAOS-2). We found that SAOS-2 fusion restored CD44 expression in C33A cells, suggesting that a trans-acting factor present in SAOS-2 cells promotes CD44 production. C33A cells are BRG-1-deficient, and we found that CD44 was absent in another BRG-1-deficient tumor cell line, indicating that loss of BRG-1 may be a general mechanism by which cells lose CD44. Reintroduction of BRG-1 into these cells restored CD44 expression. Furthermore, disruption of BRG-1 function through the use of dominant-negative BRG-1 demonstrated the requirement of BRG-1 in CD44 regulation. Finally, we show that Cyclin E overexpression resulted in the attenuation of CD44 stimulation, which is consistent with previous observations that Cyclin E can abrogate BRG-1 action. Taken together, these results suggest that BRG-1 is a critical regulator of CD44 expression, thus implicating SWI/SNF components in the regulation of cellular adhesion and metastasis.

Mobilization kinetics of CD34(+) cells in association with modulation of CD44 and CD31 expression during continuous intravenous administration of G-CSF in normal donors.

The aim of the present study is to evaluate the kinetics of CD34(+) cells and investigate the potential modulation of CD44 and CD31 expression on CD34(+) cells during continuous i.v. administration of G-CSF, thus to elucidate the possible mechanism of peripheral blood progenitor cell (PBPC) mobilization. Fifteen healthy donors were enrolled in this study. G-CSF (10 microg/kg/day) was administered for four consecutive days through continuous 24-h i.v. infusion. For measurement of complete blood counts, CD34(+) cell levels and their expression of CD44 and CD31, PB sampling was performed immediately before the administration of G-CSF (steady-state) and after 4, 8, 24, 48, 72, 96, and 120 h of G-CSF administration. The percentage and absolute number of CD34(+) cells significantly increased at day 3 (0. 55 +/- 0.09%, 51.12 +/- 24.83 x 10(3)/ml) and day 4 (0.47 +/- 0.09%, 46.66 +/- 24.93 x 10(3)/ml), compared to the steady-state level (0. 06 +/- 0.09%, 2.03 +/- 5.69 x 10(3)/ml). At day 3 to day 5 following the onset of G-CSF administration, a strong decrease of CD44 and CD31 expression was observed on mobilized CD34(+) cells compared to controls: the relative fluorescence intensity of CD44 and CD31 was, respectively, 50%-70% and 40%-90% lower than that of controls. We conclude that continuous i.v. administration of G-CSF apparently results in more rapid mobilization of CD34(+) cells, and downregulation of CD44 and CD31 on CD34(+) cells is likely to be involved in the mobilization of PBPC after treatment with G-CSF.

CD44s expression in human colon carcinomas influences growth of liver metastases.

CD44 is a family of cell-surface adhesion molecules which exist in several isoforms arising from mRNA alternative. Malignant transformation of colonic mucosa is associated with alterations in CD44 expression, which result in up-regulation of high-molecular-weight CD44 isoforms and down-regulation of CD44s. We have demonstrated that stable transfection of CD44s into colon-carcinoma cell lines reduces their tumorigenicity. To understand the influence of CD44s expression on the metastatic potential of human colon carcinomas, we measured the ability of several different CD44s-transfected colon carcinomas to establish experimental liver metastases following splenic inoculation in mice. We observed that introduction of CD44s into 2 different human colon carcinoma cell lines, HT29 and KM12C6, resulted in reduced growth of liver metastases by as much as 75%. To explore the relationship between hyaluronate adhesion and metastasis, we transfected HT29 cells with cDNA encoding a mutant CD44s that does not bind to hyaluronate. HT29 transfectants expressing this mutant CD44s demonstrate an 84% reduction in growth of liver metastases, despite minimal binding to hyaluronate by the mutant CD44s. In concert, these results indicate that CD44s down-regulation, which occurs with malignant transformation of colonic mucosa, is associated with enhanced growth of experimental liver metastases. Consequently, the functional consequences of CD44s down-regulation in colon carcinomas may be just as significant as the consequences of up-regulation of other CD44 isoforms.

Introduction of antisense CD44s cDNA down-regulates expression of overall CD44 isoforms and inhibits tumor growth and metastasis in highly metastatic colon carcinoma cells

We created antisense CD44 transfectants using LS174T, a colon adenocarcinoma cell line and assessed the effects of overall CD44 down-regulation on colorectal tumor growth and metastasis. The expression of antisense CD44s (the standard form of CD44) cDNA markedly inhibited the overall expression of CD44 variants. In vitro studies showed a significantly reduced ability of the stable antisense transfectants (LS174TAS1 and LS174TAS2) to bind hyaluronate and osteopontin, ligands for CD44. These cells developed tumors more slowly than controls (parental LS174T and mock transfectants) when the cells were subcutaneously injected into SCID mice. However, in vitro proliferation assays demonstrated no significant difference between the antisense transfectants and the controls on a hyaluronate-coated surface, suggesting the participation of ligands other than hyaluronate in tumor growth in vivo. Intrasplenic injection of parental LS174T cells produced colonies in the liver in 10 of 11 mice, whereas mice injected with the antisense transfectants were completely free of metastasis. In peritoneal dissemination, the weight of nodules and amount of ascites were significantly reduced in LS174TAS1 and AS2 compared with the controls. In vitro adhesion assays between the transfectants or controls and human peritoneal mesothelial cells revealed that the binding of LS174T cells to mesothelial cells was partly mediated by CD44-hyaluronate interaction. These data suggest that CD44-hyaluronate interaction plays a crucial role in peritoneal dissemination in colorectal carcinoma. The results of our study demonstrate the possible application of antisense CD44s to the treatment of colorectal carcinoma.

CD44 expression marks the onset of keratinocyte stratification and mesenchymal maturation into fibrous dermis in fetal human skin.

The cell surface glycoprotein CD44 is involved in active cell movement, cancer metastasis, and morphogenesis. We studied its expression in fetal human skin using an antibody specific for CD44v3 and another that recognizes all CD44 forms. In embryonic and early fetal skin, only cells with dendritic morphology expressed CD44. The first keratinocyte expression of CD44 occurred in the basal cells on the eleventh week. Later, the suprabasal cells also turned positive, whereas periderm and the terminally differentiated cells remained negative at all stages. Maturation of the early mesenchyme towards dermis at the eleventh week was associated with an increase in the number of CD44-positive cells, and later the fibrous extracellular matrix also became CD44-positive. During hair induction, the epithelium showed a transient downregulation of CD44. Later, the follicular cells regained CD44. Cells in the primordial dermal papilla displayed a continuously strong signal. The sweat gland anlagen showed faint CD44 positivity. Exon 3 was present in the CD44 of keratinocytes and their derivatives but was absent in dermis. CD44 expression in human fetal skin is a relatively late event, associated with maturation and adult-type differentiation both in epidermal keratinocytes and in dermal fibroblastic cells.(J Histochem Cytochem 47:1617-1624, 1999)

Distinct patterns of apoptosis in association with modulation of CD44 induced by thrombopoietin and granulocyte-colony stimulating factor during ex vivo expansion of human cord blood CD34+ cells.

The insufficient number of haemopoietic stem cells (HSCs) in cord blood (CB) is the major potential limitation to widespread use of CB for marrow replacement. Cytokine-mediated ex vivo expansion has been proposed as a means of increasing the number of CB HSCs for transplantation. However, the biology of CB HSCs during cytokine-mediated ex vivo expansion, such as apoptosis or expression of adhesion molecules, has not yet been elucidated. We have investigated the patterns of apoptosis and CD44 expression on human CB CD34+ cells during ex vivo expansion. CD34+ cells isolated from human CB were cultured in a stroma-free liquid culture system with thrombopoietin (TPO), flt3-ligand (FL), stem cell factor (SCF), and/or granulocyte-colony stimulating factor (G-CSF). During the culture, for up to 5 weeks, apoptosis was measured by staining with 7-amino-actinomycin D (7-AAD) along with concurrent immunophenotyping of CD34 and CD44 with three-colour flow cytometry. In the cultures with TPO, an apoptotic fraction with down-regulated CD44 appeared from the fourth day up to the second week. G-CSF also induced apoptosis but in a different manner; the apoptotic fraction without down-regulation of CD44 appeared unremittingly for up to 5 weeks. FL did not induce apoptosis or down-regulation of CD44. These findings show that apoptosis is indeed involved in the regulation of CB CD34+ cells in ex vivo expansion and the patterns of apoptosis are dependent on the type of cytokines used. The distinct patterns of apoptosis suggest different mechanisms of TPO and G-CSF in inducing apoptosis, which still remains to be elucidated.

Interferon-γ Inhibits CD44–Hyaluronan Interactions in Normal Human B Lymphocytes

The interaction of CD44 with its ligand hyaluronan (HA) plays a vital role in lymphopoiesis, lymphocyte homing, T cell activation, and metastasis. This study addresses the effect of cytokines involved in B cell growth on CD44–HA interactions in normal human B cells. Activation of B lymphocytes with LPS, pokeweed mitogen, or anti-IgM antibodies with or without IL-2 or IL-4 failed to induce HA adhesion. Stimulation of B cells with the phorbol ester PMA, however, induced strong HA recognition, which was inhibited by IFN-γ and to some extent by IL-4. Investigation of the potential molecular mechanism involved revealed that PMA-induced HA adhesion correlated with enhanced expression of CD44-H- and V6-containing isoforms, as determined by flow cytometry, and the differential induction of V4- and V5-containing isoforms, as determined by reverse transcriptase-based polymerase chain reaction analysis. The inhibition of PMA-induced adhesion by IFN-γ and IL-4 correlated with the downregulation of CD44 H expression and altered usage of exons V4 and V5. However, changes in the electrophoretic mobility of CD44 proteins, as a measure of posttranslational modifications, were not detected in response to PMA and IFN-γ or PMA and IL-4. These results suggest that the inhibition of PMA-induced HA adhesion by IFN-γ and IL-4 may influence B cell migration through their ability to downregulate CD44–HA interactions.

Down-regulation of CD44 expression in human prostatic carcinoma cell lines is correlated with DNA hypermethylation.

Down-regulation of the cell-surface adhesion molecule CD44 has been suggested to play an important role in tumor progression and metastasis of prostate cancer. CD44 is encoded by a gene that contains a CpG-rich region (CpG island) in its 5' regulatory sequence. We tried to assess whether hypermethylation of this region is the mechanism responsible for CD44 transcriptional inactivation. A panel of prostatic-carcinoma cell lines, Du145, LNCaP, PC3, PC346C and TSU, was analyzed for CD44 mRNA and protein expression. Du145, PC3 and TSU were positive for CD44, whereas in LNCaP and PC346C both CD44 mRNA and protein expression was suppressed. Methylation-sensitive restriction-enzyme analysis of genomic DNA showed that, in contrast to the CD44-positive cell lines, the CD44-negative lines were hypermethylated in the CD44 promoter CpG island. Furthermore, treatment of a PC346C culture with the demethylating agent 5-azacytidine resulted in re-expression of CD44 mRNA. It is concluded that hypermethylation of the CD44 5' promoter region is one of the mechanisms by which CD44 expression is down-regulated in prostatic-carcinoma cell lines.

CD44 and CD44v6 downregulation in clinical prostatic carcinoma: relation to Gleason grade and cytoarchitecture.

Altered expression of CD44 has been implicated in tumor progression and metastasis in multiple neoplasms. CD44 expression in archival tissues of prostate carcinoma was examined by immunohistochemistry with monoclonal antibodies against core CD44 and the RNA splice variant CD44v6 (v6). Core CD44 expression was reduced in the majority of primary neoplastic foci (n = 94) and loss of expression correlated with increasing Gleason grade. Staining for v6 was absent in most carcinomas and metastases. Expression of core CD44 in pelvic lymph node (n = 27) and bone metastases (n = 21) was significantly reduced. In addition, CD44 expression correlated with cytoarchitecture. Tall columnar tumor cells typically stained positively, yet more rounded cells forming cribiform structures or nests showed reduced expression. All cases of high-grade prostatic intraepithelial neoplasia were positive for core CD44 yet, there was decreased expression in cribiform and micropapillary variants. The majority of clinically relevant human prostatic carcinomas and metastases downregulate expression of CD44. Additional studies to determine whether CD44 cell surface expression relates to clinical outcome independent of other established clinicopathologic risk factors are warranted.

CD44 and CD44v6 downregulation in clinical prostatic carcinoma: Relation to Gleason grade and cytoarchitecture

BACKGROUND Altered expression of CD44 has been implicated in tumor progression and metastasis in multiple neoplasms. METHODS CD44 expression in archival tissues of prostate carcinoma was examined by immunohistochemistry with monoclonal antibodies against core CD44 and the RNA splice variant CD44v6 (v6). RESULTS Core CD44 expression was reduced in the majority of primary neoplastic foci (n = 94) and loss of expression correlated with increasing Gleason grade. Staining for v6 was absent in most carcinomas and metastases. Expression of core CD44 in pelvic lymph node (n = 27) and bone metastases (n = 21) was significantly reduced. In addition, CD44 expression correlated with cytoarchitecture. Tall columnar tumor cells typically stained positively, yet more rounded cells forming cribiform structures or nests showed reduced expression. All cases of high-grade prostatic intraepithelial neoplasia were positive for core CD44 yet, there was decreased expression in cribiform and micropapillary variants. CONCLUSIONS The majority of clinically relevant human prostatic carcinomas and metastases downregulate expression of CD44. Additional studies to determine whether CD44 cell surface expression relates to clinical outcome independent of other established clinicopathologic risk factors are warranted. Prostate 34:162–168, 1998.© 1998 Wiley-Liss, Inc.

CD44 as a marker in human cancers.

Tumor metastasis is one of the most life-threatening aspects of tumor progression in patients with cancer. One of the cell surface molecules that has been implicated to play an important role in tumor metastasis is CD44. Earlier results provide the initial optimism that CD44 isoform expression may be a marker for human cancers. However, more recent studies revealed that regulation of CD44 isoform expression is a complex and not well-understood phenomenon. Expression of CD44 in tumor cells can be regulated quantitatively by increasing the expression of one particular CD44 isoform or quantitatively by altering the expression of CD44 isoforms. Downregulation of CD44 is important in the metastasis of some tumor cells. We summarize some of the recent results on the potential of CD44 as a diagnostic or prognostic marker for patients with cancers.

Expression of β7 Integrins and Other Cell Adhesion Molecules on Mouse Lymphocytes and Their Modulation by a New Cytokine, IL-2 Receptor-Inducing Factor

We explored the role of a new cytokine, IL-2 receptor-inducing factor (IL-2RIF), in intestinal mucosai immunity and in the regulation of integrin β7 receptors on intestinal lymphocytes. Most SIEL (small intestine intraepithelial lymphocytes) were M290 (αM290β7) positive, while only 10 to 15% of SIEL were R1-2 (α4) positive. The expression of α4 (R1-2) and β7 (M293) but not αM290β7 integrin on SIEL was up-regulated by IL-2RIF. Incubating SIEL with IL-2RIF resulted in the up-regulation of CD45RB and down-regulation of CD44. About 50% of LPL (lamina propria lymphocytes) were αM290β7 positive, while only 20% of LPL were α4 positive. The expression of αM290β7 integrin on LPL was down-regulated and α4 and β7 integrin was up-regulated by IL-2RIF. Incubating LPL with IL-2RIF resulted in the up-regulation of CD44 and no significant change of CD11a, CD45, CD45RB, and ICAM-1. These results suggested that SIEL and LPL may play a different role in intestinal mucosal immunity and that IL-2RIF may play an important role in regulating the functions of integrins β7 on IEL and LPL.

A novel form of self tolerance dictated in the thymus of transgenic mice with autoreactive TCR alpha and beta chain genes.

Transgenic (TG) mice with TCR alpha and beta chain genes from a CD4-dependent auto-I-Ak reactive T cell clone were generated. H-2k TG mice had a large number of thymic and splenic CD4 T cells expressing the autoreactive TCR without manifestation of autoimmunity. The cells were not anergic, as they could respond to autologous antigen presenting cells and anti-TCR antibodies in vitro to proliferate and to produce interleukins. Various degrees of down-regulation of CD2 and CD44 was observed in TG mice, indicating the presence of a defective co-stimulatory process in TG T cells. These features indicate that the self tolerance in autoreactive TCR TG mice is due not to clonal deletion and anergy but to a novel mechanism where T cells cannot sufficiently respond to normally existing self ligand in vivo. That such an in vivo unresponsiveness of autoreactive T cells is dictated in the thymus during CD4 T cell differentiation atypical form of positive selection of autoreactive T cells was suggested by the abnormal surface expression of CD69 and HSA.

Expression of CD44 is repressed in neuroblastoma cells.

We have used cDNA subtractive cloning to identify a group of human genes that are expressed in diverse differentiated derivatives of neural crest origin but not in neuroblastoma cell lines. One of these genes was identified as CD44, which encodes an integral membrane glycoprotein that serves as the principal receptor for hyaluronate and participates in specific cell-cell and cell-extracellular matrix interactions. The repression of CD44 expression in neuroblastoma cell lines might be relevant to their high metastatic potential. We have cloned full-length cDNAs corresponding to CD44 trancscripts and identified a novel splice variant of CD44 lacking 31 amino acids of the extracellular domain. As a first step toward analysis of CD44 downregulation in neuroblastoma cells, we have mapped the CD44 RNA initiation site and analyzed the structure of the upstream regulatory region. We constructed a series of plasmids containing different amounts of CD44 upstream regulatory region linked to the bacterial chloramphenicol acetyltransferase gene and then analyzed their ability to promote transcription in neuroblastoma and melanoma cells. We found that a DNA segment including about 150 bp of the CD44 upstream region and the 5' end of the gene itself was sufficient to induce substantial transcription of the chloramphenicol acetyltransferase gene in both neuroblastoma and melanoma cells. Several upstream cis-acting elements contribute to the downregulation of CD44 in neuroblastoma cells, the most prominent being a 120-bp DNA fragment located 450 bp upstream to the RNA initiation site. Our data suggest that multiple factors might be involved in downregulation of CD44 in neuroblastoma cells.

Expression of CD44 Is Repressed in Neuroblastoma Cells

We have used cDNA subtractive cloning to identify a group of human genes that are expressed in diverse differentiated derivatives of neural crest origin but not in neuroblastoma cell lines. One of these genes was identified as CD44, which encodes an integral membrane glycoprotein that serves as the principal receptor for hyaluronate and participates in specific cell-cell and cell-extracellular matrix interactions. The repression of CD44 expression in neuroblastoma cell lines might be relevant to their high metastatic potential. We have cloned full-length cDNAs corresponding to CD44 trancscripts and identified a novel splice variant of CD44 lacking 31 amino acids of the extracellular domain. As a first step toward analysis of CD44 downregulation in neuroblastoma cells, we have mapped the CD44 RNA initiation site and analyzed the structure of the upstream regulatory region. We constructed a series of plasmids containing different amounts of CD44 upstream regulatory region linked to the bacterial chloramphenicol acetyltransferase gene and then analyzed their ability to promote transcription in neuroblastoma and melanoma cells. We found that a DNA segment including about 150 bp of the CD44 upstream region and the 5' end of the gene itself was sufficient to induce substantial transcription of the chloramphenicol acetyltransferase gene in both neuroblastoma and melanoma cells. Several upstream cis-acting elements contribute to the downregulation of CD44 in neuroblastoma cells, the most prominent being a 120-bp DNA fragment located 450 bp upstream to the RNA initiation site. Our data suggest that multiple factors might be involved in downregulation of CD44 in neuroblastoma cells.

Expression of the CD11/CD18, leukocyte adhesion molecule 1, and CD44 adhesion molecules during normal myeloid and erythroid differentiation in humans

We have used three-color flow cytometry to investigate the pattern of expression of the CD11/CD18, CD44, and leukocyte adhesion molecule 1 (LAM-1) adhesion molecules during myeloid and erythroid differentiation in humans. The earliest myeloid cells, identified as CD33loCD15-, were exclusively CD44hi but contained both leukocyte function-associated antigen 1 (LFA-1hi) and LFA-1lo cells, as well as LAM-1+ and LAM-1- cells. This CD33loCD15- myeloid subpopulation expressed only low levels of CD11c and failed to express CD11b, CD14, or any lymphoid (CD3, CD16, CD19) antigens or glycophorin. Commitment to monocyte differentiation, suggested by the presence of an LFA-1hi CD11c+ subset within the CD33loCD15- subpopulation, was clearly signaled by upregulation of CD33; these monocyte-lineage committed cells were exclusively CD33hi, CD44hi, CD11ahi, CD11c+, and exhibited a broad range of intensity of CD15 expression. Later stages of monopoiesis were identified by acquisition of CD11b, and subsequently of CD14. Myeloid cells committed to granulopoiesis remained LFA-1lo, and underwent a sharp upregulation of CD15 along with downregulation of both CD33 and CD44. Successive stages of granulocyte development were marked by expression of CD11b and, subsequently, of CD16. The earliest cells capable of erythroid differentiation were CD44hi, LFA-1lo, and LAM-1+. Both LFA-1 and LAM-1 were lost before the onset of glycophorin (glyco) expression, whereas CD44 expression remained high on glyco+ cells, which also expressed CD45. CD44 expression was intermediate on glyco+ CD71+ cells, and low on glyco+ CD45- CD71- cells, similar to normal, circulating erythrocytes. Our results allow us to phenotypically define discrete stages in the normal development of monocytes, neutrophils, and erythrocytes. The expression of LFA-1, LAM-1, and high levels of CD44 on the most primitive hematopoietic cells detectable by flow cytometry suggests that at least some of these molecules are critically involved in leukocyte adhesion during development.