SOD3 Expression in Tumor Stroma Provides the Tumor Vessel Maturity in Oral Squamous Cell Carcinoma.

Tumor angiogenesis is one of the hallmarks of solid tumor development. The progressive tumor cells produce the angiogenic factors and promote tumor angiogenesis. However, how the tumor stromal cells influence tumor vascularization is still unclear. In the present study, we evaluated the effects of oral squamous cell carcinoma (OSCC) stromal cells on tumor vascularization. The tumor stromal cells were isolated from two OSCC patients with different subtypes: low invasive verrucous squamous carcinoma (VSCC) and highly invasive squamous cell carcinoma (SCC) and co-xenografted with the human OSCC cell line (HSC-2) on nude mice. In comparison, the CD34+ vessels in HSC-2+VSCC were larger than in HSC-2+SCC. Interestingly, the vessels in the HSC-2+VSCC expressed vascular endothelial cadherin (VE-cadherin), indicating well-formed vascularization. Our microarray data revealed that the expression of extracellular superoxide dismutase, SOD3 mRNA is higher in VSCC stromal cells than in SCC stromal cells. Moreover, we observed that SOD3 colocalized with VE-cadherin on endothelial cells of low invasive stroma xenograft. These data suggested that SOD3 expression in stromal cells may potentially regulate tumor vascularization in OSCC. Thus, our study suggests the potential interest in SOD3-related vascular integrity for a better OSCC therapeutic strategy.

Investigation of bone invasion and underlying mechanisms of oral cancer using a cell line-derived xenograft model.

The cancer stroma regulates bone invasion in oral squamous cell carcinoma (OSCC). However, data on normal stroma are limited. In the present study, the effects of gingival and periodontal ligament tissue-derived stromal cells (G-SCs and P-SCs, respectively) and human dermal fibroblasts (HDFs) on bone resorption and osteoclast activation were assessed using hematoxylin and eosin and tartrate-resistant acid phosphatase staining in a cell line-derived xenograft model. The results demonstrated that G-SCs promoted bone invasion and osteoclast activation and inhibited osteoclast proliferation following crosstalk with the human OSCC HSC-3 cell line, whereas P-SCs inhibited bone resorption and promoted osteoclast proliferation in vitro but had a minimal effect on osteoclast activation both in vitro and in vivo following crosstalk with HSC-3 cells. Furthermore, the effects of G-SCs, P-SCs and HDFs on protein expression levels of matrix metalloproteinase (MMP)-9, membrane type 1 MMP (MT1-MMP), Snail, parathyroid hormone-related peptide (PTHrP) and receptor activator of NF-κB ligand (RANKL) in HSC-3 cells in OSCC bone invasion regions were assessed using immunohistochemistry. The results demonstrated that G-SCs had a more prominent effect on the expression of MMP-9, MT1-MMP, Snail, PTHrP, and RANKL, whereas P-SCs only promoted RANKL and PTHrP expression and exerted a minimal effect on MMP-9, MT1-MMP and Snail expression. The potential genes underlying the differential effects of G-SCs and P-SCs on bone invasion in OSCC were evaluated using a microarray, which indicated that cyclin-dependent kinase 1, insulin, aurora kinase A, cyclin B1 and DNA topoisomerase II alpha underlaid these differential effects. Therefore, these results demonstrated that G-SCs promoted bone invasion in OSCC by activating osteoclasts on the bone surface, whereas P-SCs exerted an inhibitory effect. These findings could indicate a potential regulatory mechanism for bone invasion in OSCC.

Crosstalk between cancer and different cancer stroma subtypes promotes the infiltration of tumor­associated macrophages into the tumor microenvironment of oral squamous cell carcinoma.

Tumor­associated macrophages (TAMs) are linked to the progression of numerous types of cancer. However, the effects of the tumor microenvironment (TME) of oral squamous cell carcinoma (OSCC), particularly the cancer stroma on TAMs, remains to be elucidated. In the present study, the effects of verrucous SCC­associated stromal cells (VSCC­SCs), SCC­associated stromal cells (SCC­SCs) and human dermal fibroblasts (HDFs) on the differentiation, proliferation and migration of macrophages in vitro was assayed using Giemsa staining, and immunofluorescence, MTS and Transwell (migration) assays, respectively. The combined results suggested that both VSCC­SCs and SCC­SCs promoted the differentiation of macrophages into M2 type TAMs, as well as the proliferation and migration of macrophages following crosstalk with HSC­3 cells in vitro. Moreover, the SCC­SCs exerted a more prominent effect on TAMs than the VSCC­SCs. Immunohistochemical staining was used to examine the expression of CD34, CD45, CD11b and CD163 to assay the effects of VSCC­SCs, SCC­SCs and HDFs on microvessel density (MVD) and the infiltration of CD45(+) monocytes, CD11b(+) TAMs and CD163(+) M2 type macrophages. The results suggested that both VSCC­SCs and SCC­SCs promoted MVD and the infiltration of CD45(+) monocytes, CD11b(+) TAMs and CD163(+) M2 type TAMs into the TME of OSCC following crosstalk with HSC­3 cells in vivo. The SCC­SCs exerted a more prominent promoting effect than the VSCC­SCs. Finally, the potential genes underlying the differential effects of VSCC­SCs and SCC­SCs on the infiltration of TAMs were investigated using microarray analysis. The results revealed that interleukin 1ß, bone morphogenetic protein 4, interleukin 6 and C­X­C motif chemokine ligand 12 had great potential to mediate the differential effects of VSCC­SCs and SCC­SCs on TAM infiltration. On the whole, the findings presented herein, demonstrate that both VSCC­SCs and SCC­SCs promote the infiltration of TAMs into the TME of OSCC following crosstalk with HSC­3 cells; the SCC­SCs were found to exert a more prominent promoting effect. This may represent a potential regulatory mechanism for the infiltration of TAMs into the TME of OSCC.

Differentiation and roles of bone marrow-derived cells on the tumor microenvironment of oral squamous cell carcinoma.

The stroma affects the properties and dynamics of the tumor. Previous studies have demonstrated that bone marrow-derived cells (BMDCs) possess the capability of differentiating into stromal cells. However, the characteristics and roles of BMDCs in oral squamous cell carcinoma remain unclear. The current study therefore investigated their locations and features by tracing green fluorescent protein (GFP)-labeled BMDCs in a transplantation mouse model. After irradiation, BALB-c nu-nu mice were injected with bone marrow cells from C57BL/6-BALB-C-nu/nu-GFP transgenic mice. These recipient mice were then injected subcutaneously in the head with human squamous cell carcinoma-2 cells. Immunohistochemistry for GFP, Vimentin, CD11b, CD31 and α-smooth muscle actin (SMA), and double-fluorescent immunohistochemistry for GFP-Vimentin, GFP-CD11b, GFP-CD31 and GFP-α-SMA was subsequently performed. Many round-shaped GFP-positive cells were observed in the cancer stroma, which indicated that BMDCs served a predominant role in tumorigenesis. Vimentin(+) GFP(+) cells may also be a member of the cancer-associated stroma, originating from bone marrow. Round or spindle-shaped CD11b(+) GFP(+) cells identified in the present study may be macrophages derived from bone marrow. CD31(+)GFP(+) cells exhibited a high tendency towards bone marrow-derived angioblasts. The results also indicated that spindle-shaped α-SMA(+) GFP(+) cells were not likely to represent bone marrow-derived cancer-associated fibroblasts. BMDCs gathering within the tumor microenvironment exhibited multilineage potency and participated in several important processes, such as tumorigenesis, tumor invasion and angiogenesis.

Characterization and potential roles of bone marrow-derived stromal cells in cancer development and metastasis.

Background: The tumor microenvironment and its stromal cells play an important role in cancer development and metastasis. Bone marrow-derived cells (BMDCs), a rich source of hematopoietic and mesenchymal stem cells, putatively contribute to this tumoral stroma. However their characteristics and roles within the tumor microenvironment are unclear. In the present study, BMDCs in the tumor microenvironment were traced using the green fluorescent protein (GFP) bone marrow transplantation model. Methods: C57BL/6 mice were irradiated and rescued by bone marrow transplantation from GFP-transgenic mice. Lewis lung cancer cells were inoculated into the mice to generate subcutaneous allograft tumors or lung metastases. Confocal microscopy, immunohistochemistry for GFP, α-SMA, CD11b, CD31, CD34 and CD105, and double-fluorescent immunohistochemistry for GFP-CD11b, GFP-CD105 and GFP-CD31 were performed. Results: Round and dendritic-shaped GFP-positive mononuclear cells constituted a significant stromal subpopulation in primary tumor peripheral area (PA) and metastatic tumor area (MA) microenvironment, thus implicating an invasive and metastatic role for these cells. CD11b co-expression in GFP-positive cells suggests that round/dendritic cell subpopulations are possibly BM-derived macrophages. Identification of GFP-positive mononuclear infiltrates co-expressing CD31 suggests that these cells might be BM-derived angioblasts, whereas their non-reactivity for CD34, CD105 and α-SMA implies an altered vascular phenotype distinct from endothelial cells. Significant upregulation of GFP-positive, CD31-positive and GFP/CD31 double-positive cell densities positively correlated with PA and MA (P<0.05). Conclusion: Taken together, in vivo evidence of traceable GFP-positive BMDCs in primary and metastatic tumor microenvironment suggests that recruited BMDCs might partake in cancer invasion and metastasis, possess multilineage potency and promote angiogenesis.

The Role of Bone Marrow-Derived Cells during Ectopic Bone Formation of Mouse Femoral Muscle in GFP Mouse Bone Marrow Transplantation Model.

Multipotential ability of bone marrow-derived cells has been clarified, and their involvement in repair and maintenance of various tissues has been reported. However, the role of bone marrow-derived cells in osteogenesis remains unknown. In the present study, bone marrow-derived cells during ectopic bone formation of mouse femoral muscle were traced using a GFP bone marrow transplantation model. Bone marrow cells from C57BL/6-Tg (CAG-EGFP) mice were transplanted into C57BL/6 J wild type mice. After transplantation, insoluble bone matrix (IBM) was implanted into mouse muscle. Ectopic bone formation was histologically assessed at postoperative days 7, 14, and 28. Immunohistochemistry for GFP single staining and GFP-osteocalcin double staining was then performed. Bone marrow transplantation successfully replaced hematopoietic cells with GFP-positive donor cells. Immunohistochemical analyses revealed that osteoblasts and osteocytes involved in ectopic bone formation were GFP-negative, whereas osteoclasts and hematopoietic cells involved in bone formation were GFP-positive. These results indicate that bone marrow-derived cells might not differentiate into osteoblasts. Thus, the main role of bone marrow-derived cells in ectopic osteogenesis may not be to induce bone regeneration by differentiation into osteoblasts, but rather to contribute to microenvironment formation for bone formation by differentiating tissue stem cells into osteoblasts.

Parenchyma-stromal interactions induce fibrosis by secreting CCN2 and promote osteoclastogenesis by stimulating RANKL and CD68 through activated TGF-ß/BMP4 in ameloblastoma.

BACKGROUND: Tumor parenchyma-stromal interactions affect the properties of tumors and their dynamics. Our group previously showed that secreted frizzled related protein (sFRP)-2 impairs bone formation and promotes bone invasion in ameloblastoma. However, the effects of the secreted growth factors CCN2, TGF-ß, and BMP4 on stromal tissues in ameloblastoma remain unclear. MATERIALS AND RESULTS: Thirty-five paraffin-embedded ameloblastoma cases, ameloblastoma-derived cell lines (AM-1), and primary cultures of ameloblastoma stromal fibroblasts (ASF) were used. Immunohistochemistry, MTT assay, Western blotting, and RT-PCR were performed on these samples. Parenchyma-stromal CCN2 overexpression correlated significantly with fibrous-type stroma, but not with myxoid-type stroma, suggesting a role of CCN2 in fibrosis (P < 0.05). Recombinant CCN2 induction of enhanced ASF proliferation in AM-1 medium supports this view. Conversely, BMP4 and TGF-ß were expressed in myxoid-type fibroblasts, but little expression was found in parenchyma. RANKL-positive and CD68-positive stromal cell populations were significantly greater in myxoid-type tumor areas than in fibrous-type tumor areas, while a higher Ki-67 labeling index was recorded in ameloblastoma with fibrous-type stroma. These data suggest that stromal properties influence bone resorption-related activities and growth rates, respectively. CONCLUSIONS: These results suggest that the effects of secreted growth factors are governed by ameloblastoma parenchyma-stromal interactions. CCN2 promotes fibrogenesis independent of TGF-ß signaling. Absence of CCN2 expression is associated with a phenotypic switch to a myxoid-type microenvironment that is conducive for TGF-ß/BMP4 signaling to promote osteoclastogenesis.

Knockdown of Dkk-3 decreases cancer cell migration and invasion independently of the Wnt pathways in oral squamous cell carcinoma­derived cells.

Oral squamous cell carcinoma (OSCC) is thought to arise as the result of cumulative genetic or epigenetic alterations in cancer-associated genes. We focused on the Dickkopf-3 (Dkk-3) gene as a candidate tumor suppressor in OSCC. Dkk-3 is a potential tumor suppressor, and its downregulation has been reported in various types of malignancies. However, our previous data demonstrated that the Dkk-3 protein was dominantly expressed in OSCC tissue, and its expression was correlated with a high incidence of metastasis and with poor prognosis. In order to explain this paradox, we performed functional analyses of the Dkk-3 gene in cancer cell lines. RT-PCR revealed that Dkk-3 mRNA expression was observed in OSCC-derived cell lines but not in gastrointestinal or colorectal adenocarcinoma­derived cell lines. The siRNA for Dkk-3 was transfected into Dkk-3-expressing cells, and the changes in cell proliferation, invasion and migration were assessed. The knockdown of Dkk-3 mRNA by siRNA transfection did not affect cell proliferation, but it significantly decreased cell migration and invasion. To further investigate the precise mechanism that contributes to the potential oncogenic function of Dkk-3, the Wnt canonical pathway and non-canonical pathways were assessed. Western blotting demonstrated that the effect of Dkk-3 knockdown on cell migration or invasion was not caused by activation of the Wnt pathways. These data demonstrated that Dkk-3 expression in OSCC was different than that in adenocarcinomas. Dkk-3 may possess an oncogenic function that is independent of Wnt signaling.

Absence of Dickkopf (Dkk)-3 protein expression is correlated with longer disease-free survival and lower incidence of metastasis in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the most frequently occurring types of cancer worldwide. We focused on the fact that the aberrant function of Wnt/ß-catenin signaling is a frequent event in malignancies. Dickkopf (Dkk)-3 is a major negative regulator of Wnt/ß-catenin signaling, which is a known tumor suppressor and is down-regulated in various types of cancer. However, the expression profile of the Dkk-3 protein in HNSCC has not yet been reported. The present study was conducted to investigate Dkk-3 protein expression in 90 cases of HNSCC tissue samples and HNSCC-derived cell lines. In contrast to findings available on other types of cancer, the Western blot analysis revealed that HNSCC cell lines expressed the Dkk-3 protein. In immunohistochemistry, 76 cases (84.4%) out of 90 tissue samples were Dkk-3-positive, whereas only 14 cases (15.6%) were negative. Notably, survival analysis showed that the Dkk-3 (-) group exhibited significantly longer disease-free survival (p=0.038), metastasis-free survival (p=0.013) and longer overall survival (p=0.155). The results showed that the Dkk-3 protein was dominantly expressed and may be involved in carcinogenesis and metastasis in HNSCC. Moreover, the findings suggest that the function of Dkk-3 differs depending on the tissue of origin, and that it may exert an oncogenic function in HNSCC.

Dickkopf (Dkk)-3 and ß-catenin expressions increased in the transition from normal oral mucosal to oral squamous cell carcinoma.

Dickkopf (Dkk)-3, an inhibitor of the Wnt/ß-catenin pathway, is reported as a potential tumor suppressor gene in many cancers. To gain a better comprehension of the mechanisms involved in the carcinogenesis of oral squamous epithelium, protein expression and localization of Dkk-3 and ß-catenin was investigated in normal epithelium, dysplasias and squamous cell carcinoma (SCC). An increase in ß-catenin and Ki-67 expressions was observed from dysplasias to poorly differentiated SCC. Interestingly, an increase in Dkk-3 positive cells was also noted, which was correlated to the cancer progression step. A change in Dkk-3 localization during the transformation of normal oral epithelium to SCC was clearly observed. Dkk-3 was localized in the cell membrane in normal oral epithelium and in dysplasias, whereas that was localized in both cell membrane and cytoplasm in SCC. These results suggest that Dkk-3 is involved in the carcinogenesis of SCC with a distinct function from those in other cancers.

NRAS and BRAF mutation frequency in primary oral mucosal melanoma.

Oral mucosal melanoma (OMM) is a fatal sarcoma of unknown etiology. Histological morphology and genetic events are distinct from those of its cutaneous counterpart. Mutation and up-regulation of c-kit has been identified in OMM which may activate downstream molecules such as RAS and RAF. These molecules are involved in the mitogen-activated protein kinase (MAPK) pathway leading to tremendous cell proliferation and survival. NRAS and BRAF mutation and protein expression have been studied in other melanoma subtypes. The purpose of this study was to determine RAS protein expression and NRAS and BRAF mutation in 18 primary OMM cases using immunohistochemistry and mutation analysis. Results showed that RAS is intensely expressed in both in situ and invasive OMMs. However, NRAS mutation was only observed in 2/15 polymerase chain reaction (PCR) amplified cases both of which were silent mutations. On the other hand, BRAF missense mutations were observed only in 1/15 cases with PCR amplification. NRAS and BRAF mutations were independent from previously reported c-kit mutations. The classical V600E BRAF mutation was not found; instead a novel V600L was observed suggesting that the oncogenic event in OMM is different from that in skin melanoma. The low frequency of NRAS and BRAF mutations indicate that these genes are not common, but probable events in OMM pathogenesis, most likely independent of c-kit mutation.

Dynamic contrast-enhanced magnetic resonance imaging for estimating tumor proliferation and microvessel density of oral squamous cell carcinomas.

We evaluated the relationship between histopathological prognostic factors, tumor proliferation microvessel density (MVD), and enhancement parameters in dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in oral squamous cell carcinoma (SCC). Twenty-eight T2 and T3 patients with primary oral SCC underwent DCE-MRI using three-dimensional fast imaging with a steady-state precession sequence. Tumor cell proliferation and MVD of all surgical specimens were evaluated using immunohistochemical staining with CD34 and the antibody for proliferating cell nuclear antigen (PCNA). Regression analysis was used to statistically analyze the relationship between the PCNA labeling index or MVD and each of three DCE-MRI parameters: maximum CI (CI-max), maximum CI gain (CI-gain) and the CI-gain / CI-max ratio). The PCNA labeling index and MVD showed significant correlations with the CI-gain/CI-max ratio (P=0.0012, r=0.581 and P=0.00141, r=0.574, respectively). The assessment of DCE-MRI parameters may prove to be a valuable non-invasive method for assessing tumor cell proliferation and MVD of patients with oral cancer.

Expression and mutation analysis of epidermal growth factor receptor in head and neck squamous cell carcinoma.

The epidermal growth factor receptor (EGFR)-RAS-RAF-mitogen-activated protein kinase signaling cascade is an important pathway in cancer development and recent reports show that EGFR and its downstream signaling molecules are mutated in a number of cancers. We have analyzed 91 Japanese head and neck squamous cell carcinomas (HNSCC) and 12 HNSCC cell lines for mutations in EGFR, ErbB2, and K-ras. Exons encoding the hot-spot regions in the tyrosine kinase domain of both EGFR (exons 18, 19, and 21) and ErbB2 (exons 18-23), as well as exons 1 and 2 of K-ras were amplified by polymerase chain reaction and sequenced directly. EGFR expression was also analyzed in 65 HNSCC patients using immunohistochemistry. Only one silent mutation, C836T, was found in exon 21 of EGFR in the UT-SCC-16A cell line and its corresponding metastasic cell line UT-SCC-16B. No other mutation was found in EGFR, ErbB2, or K-ras. All tumors showed EGFR expression. In 21 (32%) tumors, EGFR was expressed weakly (+1). In 27 (42%) tumors it was expressed (+2) moderately, and in 17 (26%) tumors high expression (+3) was detected. Overexpression (+2, +3) was found in 44 tumors (68%). A worse tumor differentiation and a positive nodal stage were significantly associated with EGFR overexpression (P = 0.02, P = 0.032, respectively). Similar to patients from western ethnicity, mutations are absent or rare in Japanese HNSCC. Protein overexpression rather than mutation might be responsible for activation of the EGFR pathway in HNSCC.

Cell-type specific occurrence of apoptosis in taste buds of the rat circumvallate papilla.

The present study employed immunohistochemistry for single-stranded DNA (ssDNA) to detect apoptotic cells in taste buds of the rat circumvallate papilla. Double-labeling of ssDNA and markers for each cell type - phospholipase C beta2 (PLCbeta2) and alpha-gustducin for type II cells, neural cell adhesion molecule (NCAM) for type III cells, and Jacalin for type IV cells - was also performed to reveal which types of cells die by apoptosis. We detected approximately 16.8% and 14.0% of ssDNA-immunoreactive nuclei among PLCbeta2-immunoreactive and alpha-gustducinimmunoreactive cells, respectively, but rarely found ssDNA-immunoreactive cells among NCAM-immunoreactive or Jacalin-labeled cells, indicating that type II cells die by apoptosis. We also applied double labeling of ssDNA and human blood group antigen H (AbH) - which mostly labels type I cells as well as other cell types - and found that approximately 78% of ssDNA-immunoreactive cells were labeled with AbH, indicating that apoptosis also occurs in type I cells. The present results revealed that apoptosis occurs in both type I cells (dark cells) and type II cells (light cells), suggesting that there are two major cell lineages (dark cell and light cell lineages) for the differentiation of taste bud cells. In summury, type IV cells differentiate into dark and light cells and type III cells differentiate to type II cells within the light cell line.

Jacalin and peanut agglutinin (PNA) bindings in the taste bud cells of the rat: new reliable markers for type IV cells of the rat taste buds.

Lectin histochemistry of Jacalin (Artocarpus integrifolia) and peanut agglutinin (PNA), specific lectins for galactosyl (beta-1, 3) N-acetylgalactosamine (galactosyl (beta-1, 3) GalNAc), was applied to the gustatory epithelium of the adult rat. In the ordinary lingual epithelium, Jacalin and PNA labeled the cell membrane from the basal to granular cell layer. They also bound membranes of rounded-cells at the basal portion of taste buds, but the number of PNA labeled cells was smaller than that of Jacalin labeled cells. There was no apparent difference in the binding patterns of Jacalin and PNA among the taste buds of the lingual papillae and those of the palatal epithelium. Occasionally, a few spindle-shaped cells were labeled with Jacalin, but not with PNA. Double labeling of Jacalin and alpha-gustducin, a specific marker for type II cells, revealed that Jacalin-labeled spindle-shaped taste cells were immunonegative for alpha-gustducin. Spindle-shaped cells expressing protein gene product 9.5 (PGP 9.5) immunoreactivity lacked Jacalin labeling. During the development of taste buds in circumvallate papillae, the binding pattern of Jacalin became almost identical from postnatal day 5. The present results indicate that rounded cells at the basal portion of the taste buds cells (type IV cells) bind to Jacalin and PNA, and these lectins are specific markers for type IV cells of the rat taste cells.

Localization of Ulex europaeus agglutinin-I (UEA-I) in the developing gustatory epithelium of the rat.

To understand the development of the gustatory structures necessitates a reliable marker for both immature and mature taste buds. It has been reported that the intragemmal cells within the taste buds of adult rats were bound to Ulex europaeus agglutinin-I (UEA-I), a specific lectin for alpha-linked fucose, but it has not been determined whether immature taste buds, i.e. taste buds without an apparent taste pore, are labeled with UEA-I. The present study was conducted to examine the UEA-I binding pattern during the development of the rat gustatory epithelium. In adult animals, UEA-I bound to the membrane of taste buds in all examined regions of the gustatory epithelium. Within the individual taste buds, UEA-I labeled almost all intragemmal cells. The binding of UEA-I was occasionally detected below the keratinized layer of the trench wall epithelium but could not be found in the lingual epithelium of the adult animal. During the development of circumvallate papilla, some cells within the immature taste buds were also labeled with UEA-I. The developmental changes in the UEA-I binding pattern in fungiform papillae were almost identical to those in the circumvallate papilla: both immature and mature taste buds were labeled with UEA-I. The present results indicate that UEA-I is a specific lectin for the intragemmal cells of both immature and mature taste buds and, thus, UEA-I can be used as a reliable marker for all taste buds in the rat.

Human blood group antigen H is not the specific marker for type I cells in the taste buds.

We examined the localization of human blood antigen H (AbH) and its correlation with other cell type markers in the taste buds of circumvallate papillae of the adult rat. Immunoreactivity for AbH was localized in the membrane of two cell populations in the taste buds: in spindle-shaped cells extending from base to the apical portion of the taste buds as well as in round-shaped cells at the basal portion of the taste buds. Quantitative analysis revealed that approximately 47.8%, 24.4%, and 14.6% of cells within the taste buds displayed AbH-, alpha-gustducin- or protein gene product 9.5 (PGP 9.5)-immunoreactivity, respectively. Approximately 16.3% and 6.6% of AbH-immunoreactive taste bud cells displayed alpha-gustducin- or PGP 9.5-immunoreactivity, respectively. Although previous studies proposed that AbH immunoreactivity was specific for type I cells (dark cells or supporting cells), the present results indicate that AbH immunoreactivity is also present in some type II cells (alpha-gustducin immunoreactive cells) and type III cells (PGP 9.5-immunoreactive cells).