CD44s and CD44v6 expression in head and neck epithelia

Background

CD44 splice variants are long-known as being associated with cell transformation. Recently, the standard form of CD44 (CD44s) was shown to be part of the signature of cancer stem cells (CSCs) in colon, breast, and in head and neck squamous cell carcinomas (HNSCC). This is somewhat in contradiction to previous reports on the expression of CD44s in HNSCC. The aim of the present study was to clarify the actual pattern of CD44 expression in head and neck epithelia.

Methods

Expression of CD44s and CD44v6 was analysed by immunohistochemistry with specific antibodies in primary head and neck tissues. Scoring of all specimens followed a two-parameters system, which implemented percentages of positive cells and staining intensities from − to +++ (score = %×intensity; resulting max. score 300). In addition, cell surface expression of CD44s and CD44v6 was assessed in lymphocytes and HNSCC.

Results

In normal epithelia CD44s and CD44v6 were expressed in 60–95% and 50–80% of cells and yielded mean scores with a standard error of a mean (SEM) of 249.5±14.5 and 198±11.13, respectively. In oral leukoplakia and in moderately differentiated carcinomas CD44s and CD44v6 levels were slightly increased (278.9±7.16 and 242±11.7; 291.8±5.88 and 287.3±6.88). Carcinomas in situ displayed unchanged levels of both proteins whereas poorly differentiated carcinomas consistently expressed diminished CD44s and CD44v6 levels. Lymphocytes and HNSCC lines strongly expressed CD44s but not CD44v6.

Conclusion

CD44s and CD44v6 expression does not distinguish normal from benign or malignant epithelia of the head and neck. CD44s and CD44v6 were abundantly present in the great majority of cells in head and neck tissues, including carcinomas. Hence, the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision.     

CD44v10 expression in the mouse and functional activity in delayed type hypersensitivity

We have described recently that expression of CD44 exon v10 (CD44v10) is down-regulated upon metastasis of squamous cell carcinoma, whereas it is up-regulated in skin metastases of malignant melanoma. The striking regulation of CD44v10 prompted us to generate a murine CD44v10-specific monoclonal antibody to define expression and possible functions of this particular CD44 variant isoform. In the mouse, expression of exon v10 was restricted to basal layers of the epidermis and squamous epithelium of the oral cavity, the esophagus, the omasum, glandular epithelium of the submandibular and the uterine gland, as well as subpopulations of bone marrow cells and activated lymphocytes. Expression started late during development, e.g., was not observed before day 16 of gestation and there was no evidence for developmental regulation of CD44v10 expression. Functional in vivo studies revealed that anti-CD44v10 had no effect on wound healing but inhibited edema and granuloma formation in delayed type hypersensitivity (DTH). Furthermore, lymphocyte-monocyte interactions could be inhibited by anti-CD44v10. Because a CD44v10 transfected tumour line did not show any distinct pattern of cell-matrix or cell-cell adhesion, the data point toward an involvement of CD44v10 in cell migration, possibly by acting as a target structure for cytokines/chemokines provided by the contacted partner cell. J. Cell. Physiol. 171:305–317, 1997. © 1997 Wiley-Liss, Inc.     

CD44 standard and variant isoform expression in normal human skin appendages and epidermis

 CD44 isoforms have been implicated in tumor progression and metastasis formation. This study presents a thorough immunohistochemical analysis of CD44 standard and isoform expression in normal human skin appendages and epidermis applying monoclonal antibodies against CD44s, CD44v3, -v4, -v5, -v6, and -v9. An improved immunohistochemical protocol with microwave-based antigen retrieval in paraffin sections and heavy metal amplification of the diaminobenzidine reaction product provided enhanced resolution and sensitivity as compared to studies on frozen sections. The hair follicle, the seborrheic and eccrine sweat glands were strongly positive for all CD44 isoforms studied. In the latter, the clear cells but not the dark (intercalated) cells were positive. The sudoriferous ducts adjacent to the glands were weakly positive for all CD44 isoforms and strongly positive near the skin surface. In the apocrine glands, the basal cells showed only a moderate positivity. The myoepithelial cells expressed only CD44s. In the epidermis, all CD44 isoforms were detectable, with strongest CD44 immunostaining in the lower third of the stratum spinosum and weaker staining in the stratum basale and the upper two-thirds of the stratum granulosum. The stratum granulosum and corneum were unreactive. Thus, a regional and cell type-specific CD44 expression was revealed. Accepted: 10 May 1996     

Differential analysis of D-beta-Asp-containing proteins found in normal and infrared irradiated rabbit lens

Although proteins are generally composed of l-alpha-amino acids, d-beta-aspartic acid (Asp)-containing proteins have been reported in various elderly tissues. Our previous study detected several d-beta-Asp-containing proteins in a rabbit lens derived from epithelial cell line by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for d-beta-Asp-containing proteins. The identity of each spot was subsequently determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Ms-Fit online database searching algorithm. In this study, we discovered novel d-beta-Asp-containing proteins from rabbit lens. The results indicate that beta-crystallin A3, beta-crystallin A4, beta-crystallin B1, beta-crystallin B2, beta-crystallin B3, gamma-crystallin C, gamma-crystallin D, and lambda-crystallin in rabbit lens contain d-beta-Asp residues. Furthermore, the occurrence of d-beta-Asp residues increases with infrared ray (IR) irradiation. Additionally, some d-beta-Asp-containing proteins only appear after IR irradiation. One such protein is the alpha-enolase, which shows homology to tau-crystallin.     

Alternatively spliced CD44 isoforms containing exon v10 promote cellular adhesion through the recognition of chondroitin sulfate-modified CD44

Correlations have been noted between the expression of certain alternatively spliced CD44 isoforms and the metastatic propensity of various histologically distinct tumor cell types. The precise mechanism by which particular CD44 isoforms contribute to the metastatic process is, however, unclear. In the present study we demonstrate that CD44R2, a CD44 isoform highly expressed on activated and transformed hemopoietic cells, can recognize and bind a common determinant present on CD44H and CD44R1. Importantly, CD44H lacked this activity. Pretreatment of TIL1 cells expressing CD44H or CD44R1 with chondroitinase ABC inhibited adhesion to CD44R2, suggesting that the unique inserted region present within the CD44R2 molecule, encoded by exon v10, mediates cell adhesion by potentiating the recognition of chondroitin sulfate moieties presented in association with other CD44 molecules. These data help explain the differential involvement of v10-containing CD44 isoforms in tumor metastasis.     

Sulphydryl and disulphide groups in normal and irradiated epidermis.

The distribution of sulphydryl (SH) and disulphide (SS) groups in normal and irradiated epidermis of newly-born rats was studied by microspectrophotometric quantitation of the Mercury-Orange reaction. In order to obtain information on (1) the total content of both groups within the different strata of the epithelium, and (2) the mean SH and SS concentration, which is an indicator of the density of these groups per unit of tissue. The content of both groups increased in relation to the extent of irradiated area, whereas there was a decrease in the mean concentration. The increase in content of SH and SS would lead to augmented production of keratin substance previously reported in irradiated epithelia, coincidentally with an acanthotic response. The decrease in concentration of SH and SS indicates a destruction of these groups at cellular level, which may be due to membrane alterations. The findings would explain an acceleration of the keratinizing process together with modifications, both in quality and in quantity, in the horny substance.     

Coexpression of CD44 variant (v10/ex14) and CD44S in human mammary epithelial cells promotes tumorigenesis

CD44 is the major hyaluronan cell surface receptor and functions as an adhesion molecule in many different cell types, including human breast epithelial cells. The coexpression of certain CD44 variants (CD44v), such as CD44v (v10/ex14), with CD44s (standard form) appears to be closely associated with human breast tumor metastasis. In this study we have established a stable transfection of CD44v (v10/ex14) cDNA into nontumorigenic human breast epithelial cells (HBL100) which contain endogenous CD44s. Our results indicate that coexpression of both CD44v (v10/ex14) and CD44s alters the following important biological properties of these cells: 1) there is a significant reduction in hyaluronic acid (HA)-mediated cell adhesion; 2) there is an increased migration capability in collagen-matrix gel; and 3) these cells constitutively produce certain angiogenic factors and effectively promote tumorigenesis in athymic nude mice. These findings suggest that coexpression of CD44v (v10/ex14) and CD44s may trigger the onset of cell transformation required for breast cancer development. J. Cell. Physiol. 171:152–160, 1997. © 1997 Wiley-Liss, Inc.     

MOC-31和CD44v6在良恶性腹水鉴别诊断中的应用

目的探讨检测肿瘤标志物MOC-31和CD44v6对鉴别良恶性腹水的诊断价值。方法利用液基薄层细胞学自动涂片技术方法筛查出查到肿瘤细胞的恶性腹水标本390例以及良性腹水标本100例,分别采用酶联免疫吸附法(ELISA)和免疫细胞化学染色检测MOC-31和CD44v6的含量和表达情况。结果 ELISA结果显示MOC-31和CD44v6在良性腹水中的含量分别为21±4ng/ml和291±32ng/ml,在恶性腹水中的含量分别为98.1±19.3ng/ml和891±116ng/ml,差异均具有显著性(P<0.05);免疫细胞化学染色显示MOC-31在恶性腹水细胞中阳性表达250例,良性腹水细胞中阳性表达5例;CD44v6在恶性腹水细胞中阳性表达266例,良性腹水细胞中阳性表达3例,差异均具有显著性(P<0.05)。结论 MOC-31和CD44v6可以做为良恶性腹水鉴别诊断的重要指标,值得在临床病理工作中推广应用。     

大肠癌组织及癌旁组织中c—myc，COX-2和CD44v6基因表达量差异的检测

目的：通过检测大肠癌组织和癌旁组织中c-myc,COX-2以及CD44v6的表达水平,探讨这三种基因在大肠癌发生和发展中的意义。方法：应用实时荧光定量PCR技术检测了10例大肠癌组织和相应癌旁组织中c-myc,COX-2以及CD44v6基因表达水平的差异,并探讨了各基因在癌纽织中的表达水平与大肠癌临床病理指标之间的关系。结果：c-myc,COX-2以及CD44v6在大肠癌组织和癌旁组织中的表达均有非常显著性差异（P〈0．01）;癌组织中COX-2和CD44v6的表达与淋巴结转移、分化程度及Dukes分期有关（P〈0．05）。结论：c—myc,COX-2和CD44v6的异常表达均与大肠癌密切相关,三者从不同方面对大肠癌的发生和发展起到了重要作用,可作为早期诊断和预后的参考指标。     

大肠癌组织及癌旁组织中c-myc,COX-2和CD44v6基因表达量差异的检测

目的:通过检测大肠癌组织和癌旁组织中c-myc,COX-2以及CD44v6的表达水平,探讨这三种基因在大肠癌发生和发展中的意义。方法:应用实时荧光定量PCR技术检测了10例大肠癌组织和相应癌旁组织中c-myc,COX-2以及CD44v6基因表达水平的差异,并探讨了各基因在癌组织中的表达水平与大肠癌临床病理指标之间的关系。结果:c-myc,COX-2以及CD44v6在大肠癌组织和癌旁组织中的表达均有非常显著性差异(P<0.01);癌组织中COX-2和CD44v6的表达与淋巴结转移、分化程度及Dukes分期有关(P<0.05)。结论:c-myc,COX-2和CD44v6的异常表达均与大肠癌密切相关,三者从不同方面对大肠癌的发生和发展起到了重要作用,可作为早期诊断和预后的参考指标。     

Differential expression of CD44 during human prostate epithelial cell differentiation.

CD44 is a polymorphic transmembrane glycoprotein that binds hyaluronan and growth factors. Multiple isoforms of the protein can be generated by alternative splicing but little is known about the expression and function of these isoforms in normal development and differentiation. We have investigated the expression of CD44 during normal prostate epithelial cell differentiation. A conditionally immortalized prostate epithelial cell line, Pre2.8, was used as a model system. These cells proliferate at 33C but at 39C stop dividing and undergo changes consistent with early stages of cell differentiation. During the differentiation of these cells, the expression of the CD44 isoform v3-v10 was upregulated. Two layers of epithelial cells can clearly be distinguished in the human prostate, a basal layer expressing keratins 5/14 and a luminal layer expressing keratins 8/18. In prostate tissue the v3-v10 isoform was found predominantly in basal cells but also in keratin 14-negative, keratin 19-positive cells intermediate between the two layers. CD44 v3-v10 was also expressed in other keratin 14-negative prostate tissues, the ejaculatory ducts and prostatic urethra. Therefore, CD44 v3-v10 may be important as a cell surface marker for differentiating cells in the prostate epithelium.     

非小细胞肺癌中E-钙粘素和CD44v6的表达及其意义

为了探讨E-钙粘素(E-cadherin,E-cad)和白细胞分化抗原变异型6(CD44v6)在非小细胞肺癌(NSCLC)中的表达及临床意义,采用免疫组织化学(SP法)对65例NSCLC和20例癌旁组织中E-cad和CD44v6的表达进行研究。结果显示E-cad在肺癌组织中的阳性表达率(32.31%)显著低于癌旁组织(75.00%)(P<0.01),且与NSCLC的TNM分期、淋巴结转移呈负相关(P<0.05),与分化程度呈正相关(P<0.01),而与组织分型无关(P>0.05);CD44v6的阳性表达率与组织分型和分化程度无关(P>0.05),而与TNM分期和淋巴结转移呈正相关(P<0.05,<0.01)。两者在NSCLC中的表达显著相关(P<0.05)。因此检测E-cad和CD44v6的共同表达将是指导临床治疗及估计预后的有意义指标,可应用于临床预后的综合评价。     

Involvement of CD44v6 in InlB-dependent Listeria invasion

Listeria monocytogenes , a Gram-positive bacterium, is the causative agent for the disease called listeriosis. This pathogen utilizes host cell surface proteins such as E-cadherin or c-Met in order to invade eukaryotic cells. The invasion via c-Met depends on the bacterial protein InlB that activates c-Met phosphorylation and internalization mimicking in many regards HGF, the authentic c-Met ligand. In this paper, we demonstrate that the activation of c-Met induced by InlB is dependent on CD44v6, a member of the CD44 family of transmembrane glycoproteins. Inhibiting CD44v6 by means of a blocking peptide, a CD44v6 antibody or CD44v6-specific siRNA prevents the activation of c-Met induced by InlB. Subsequently, signalling, scattering and the entry of InlB-coated beads into host cells are also impaired by CD44v6 blocking reagents. For the entry process, ezrin, a protein that links the CD44v6 cytoplasmic domain to the cytoskeleton, is required as well. Most importantly, this collaboration between c-Met and CD44v6 contributes to the invasion of L. monocytogenes into target cells as demonstrated by a drastic decrease in bacterial invasion in the presence of blocking agents such as the CD44v6 peptide or antibody.     

Differential expression of ribosomal proteins in human normal and neoplastic colorectum.

Ribosomal proteins are a major component of ribosomes and play critical roles in protein biosynthesis. Recently it has been shown that the ribosomal proteins also function during various cellular processes that are independent of protein biosynthesis therefore called extraribosomal functions. In this study we have, for the first time, determined the expression profile of 12 ribosomal proteins (Sa, S8, S11, S12, S18, S24, L7, L13a, L18, L28, L32, and L35a) in normal epithelia of human colorectal mucosa using immunohistochemistry (IHC) and then compared their expression patterns with those of colorectal cancer. In the normal mucosa, ribosomal proteins were largely associated with the ribosomes of mucosal epithelia, and the expression level of ribosomal proteins, except for S11 and L7 proteins, was markedly increased in associated with maturation of the mucosal cells. On the other hand, these ribosomal proteins were markedly decreased in colorectal cancer compared with the normal mucosa. By contrast, S11 and L7 ribosomal proteins were rarely associated with the ribosomes of colorectal epithilia except immature mucosal cells, whereas their expression levels were significantly enhanced in colorectal cancer cells. In addition, L7 ribosomal protein was detected in the secretory granules of the enterochromaffin cells in the colorectal mucosa and in carcinoma cells expressing chromogranin A. These results indicate that the expression of ribosomal proteins is differentially regulated not only in normal mucosa but also in carcinoma of human colorectum, and suggest an extraribosomal function of L7 ribosomal protein in neuroendocrine function.     

结肠癌组织中CD44S和CD44V6基因蛋白的表达及意义

摘要：目的 研究CD44S和CD44V6的表达与结肠癌临床病理特征之间的关系,以明确二者在结肠癌发展和预后中的作用。方法 选取98例结肠癌组织做为实验组,采用免疫组织化学方法,检测组织中CD44S和CD44V6基因蛋白产物表达,与组织学分型、肠壁浸润深度、脉管有无浸润、淋巴结有无转移、预后等相关因素进行实验研究。结果 CD44S和CD44V6的表达与结肠癌组织类型无关,与癌组织浸润肠壁深度、脉管浸润、淋巴结转移、远处转移等密切相关。结论 CD44S和CD44V6异常表达可能参与了结肠癌的发展并可能影响患者的预后。     

Hepatocyte growth factor-induced Ras activation requires ERM proteins linked to both CD44v6 and F-actin

In several types of cells, the activation of the receptor tyrosine kinase c-Met by its ligand hepatocyte growth factor (HGF) requires the coreceptor CD44v6. The CD44 extracellular domain is necessary for c-Met autophosphorylation, whereas the intracellular domain is required for signal transduction. We have already shown that the CD44 cytoplasmic tail recruits ezrin, radixin and moesin (ERM) proteins to the complex of CD44v6, c-Met, and HGF. We have now defined the function of the ERM proteins and the step they promote in the signaling cascade. The association of ERM proteins to the coreceptor is absolutely required to mediate the HGF-dependent activation of Ras by the guanine nucleotide exchange factor Sos. The ERM proteins need, in addition, to be linked to the actin cytoskeleton to catalyze the activation of Ras. Thus, we describe here a new function of the cytoskeleton. It is part of a "signalosome" complex that organizes the activation of Ras by Sos. So far the cytoskeleton has mainly been identified as a "responder" to signal transduction. Here, we show now that F-actin acts as an "inducer" that actively organizes the signaling cascade.