Yohko Kohno

Growth and Differentiation Properties of Normal and Transformed Human Keratinocytes in Organotypic Culture

The growth and differentiation of human normal keratinocytes and their transformed counterparts were examined in organotypic cultures in which the keratinocytes were grown at the air‐liquid interface on top of contracted collagen gel containing fibroblasts. We developed a modified culture procedure including the use of a mixed medium for keratinocytes and fibroblasts. Normal keratinocytes formed a three‐dimensional structure of epithelium that closely resembled the epidermis in vivo, consisting of basal, spinous, granular and cornified layers. Cells synthesizing DNA were located in the lowest basal layer facing the collagen gel. Expressions of proteins involved in epidermal differentiation were examined by immunohistochemical staining and compared with those in skin in vivo. In the organotypic culture, transglutaminase, involucrin and filaggrin were expressed, as in the epidermis in vitro, most prominently in the granular layer. Type IV collagen, a component of basement membrane, was expressed at the interface between the keratinocyte sheet and the contracted collagen gel. Keratinocytes transformed by simian virus 40 or human papilloma virus (HPV) exhibited a highly disorganized pattern of squamous differentiation. In particular, HPV‐transformed cells invaded the collagen gel. Organotypic culture is unique in that regulatory mechanisms of growth and differentiation of keratinocytes can be investigated under conditions mimicking those in vivo.

Regulation of creatine phosphokinase B activity by protein kinase C

We previously reported that topical application of 12-o-tetradecanoylphorbol-13-acetate to mouse skin causes phosphorylation of epidermal proteins with molecular weights of 40,000 (p40) and 34,000 (p34). In the accompanying paper, p40 was identified as creatine phosphokinase B. Here we report that both in intact cells and in a cell-free system, phosphorylation of creatine hosphokinase B by protein kinase C resulted in an increase in its ability to catalyze the transfer of the high-energy phosphate of phosphocreatine to ADP, thereby producing ATP. H-7, a specific inhibitor of protein kinase C was found to abolish the increase in enzyme activity. Lineweaver-Burk plot analysis indicated that the increased activity was mostly due to a decreased Km for phosphocreatine. Phosphorylation and activation of creatine phosphokinase B may be a physiological response to maintain ATP balance when a protein kinase C pathway is stimulated.

Purification and identification of creatine phosphokinase B as a substrate of protein kinase C in mouse skin in vivo

We previously described epidermal proteins with molecular weights of 40,000 (p40) and 34,000 (p34) as target proteins of protein kinase C in mouse skin carcinogenesis in vivo. In the present work, p40 was purified from mouse brain by the use of 32P-labeled p40 of BALB/MK-2 cells as a tracer. Following four lines of evidence indicate that p40 is creatine phosphokinase B. 1) The amino acid sequences of all peptide fragments of p40 from mouse brain were located in the primary structure of creatine phosphokinase B. 2) p40 of BALB/MK-2 cells was immunoprecipitated with goat antibody against human creatine phosphokinase B. 3) p40 of BALB/MK-2 cells was absorbed to and eluted from a creatine affinity column. 4) Purified creatine phosphokinase B was phosphorylated in vitro by purified protein kinase C, but not by cAMP-dependent kinase or casein kinase II.

Bone in the marmoset: a resemblance to vitamin D-dependent rickets, type II

SummaryThe common marmoset, a New World monkey, requires a large amount of vitamin D3 to maintain its normal growth. This monkey is reported to have an end-organ resistance to 1α,25-dihydroxyvitamin D3 (1α, 25(OH)2D3). In this study, the bone morphology of marmosets fed a high vitamin D3 diet (intake of vitamin D3, 110 IU/day/100 g of body weight) was compared by X-ray and histological examinations with that of rhesus monkeys (Old World monkey) fed a normal diet (intake of vitamin D3, 5 IU/day/100 g of body weight). Three of 20 marmosets were found by X-ray examination to have osteomalacic changes in their bones despite the high daily intake of vitamin D3, whereas none of the 5 rhesus monkeys showed any signs of osteomalacica. Osteomalacic marmosets had distinct incrases in osteoid surface, relative osteoid volume, and active osteoclastic bone resorption, whereas nonosteomalacic marmosets had no increase in osteoid tissues in their bones. None of the marmosets, either osteomalacic or nonosteomalacic, was hypercalcemic despite the extremely high circulating levels of 1α,25(OH)2D3. However, the serum 25-hydroxy vitamin D3 (25OHD3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) levels were significantly lower in the osteomalacic than in the nonosteomalacic marmosets. These results suggest that the marmoset is likely to exhibit osteomalacic bone changes despite the high daily intake of vitamin D3. These changes resemble those in vitamin D-dependent rickets, type II.

DMSO induces apoptosis in SV40-transformed human keratinocytes, but not in normal keratinocytes

We found that dimethyl-sulfoxide (DMSO) at concentrations of 2.5% induced apoptosis in SV40-immortalized human keratinocytes, while normal keratinocytes were arrested at the boundary of G1/S phase under the same conditions. DMSO-induced apoptosis in SV-40 immortalized keratinocytes was not associated with change in phosphorylated state of the retinoblastoma susceptibility gene. When SV40-immortalized cells were treated with 2.5% DMSO, dissociation of the complex was observed by immunoblotting of SV40 T antigen from immunoprecipitated p53 protein fraction.

ANGPTL4 regulates the metastatic potential of oral squamous cell carcinoma.

Lymph node metastasis is a major factor for poor prognosis in oral squamous cell carcinoma (OSCC). However, the molecular mechanisms of lymph node metastasis are unclear. We determined that angiopoietin-like protein 4 (ANGPTL4) mRNA and protein expression were increased in OSCC cells established from the primary site in metastatic cases. In addition, ANGPTL4 expression in biopsy specimens was correlated with the presence of lymph node metastasis. Therefore, our initial findings suggest that OSCC cells expressing ANGPTL4 may possess metastatic ability. Furthermore, cell culture supernatants from OSCC cells that metastasized to the lymph node contain ANGPTL4 and promote invasive ability. These findings suggest that secreted ANGPTL4 may affect the invasive ability of OSCC. Moreover, the rates of positive ANGPTL4 expression at the primary site were significantly higher in the lymph node metastasis group. These results demonstrate that ANGPTL4 contributes to OSCC metastasis by stimulating cell invasion. Therefore, ANGPTL4 is a potential therapeutic target for preventing cancer metastasis.

Lin28a is a putative factor in regulating cancer stem cell-like properties in side population cells of oral squamous cell carcinoma.

Cancer stem cells (CSCs) are among the target cells of cancer therapy because they are uniquely involved in both cancer progression and sensitivity to chemotherapeutic agents. We identified side population (SP) cells, which are known to be an enriched population of CSC, in five oral squamous cell carcinoma (OSCC) cells (SCC9, SCC25, TOSCC7, TOSCC17, and TOSCC23). The percentages of SP cells ranged from 0% to 3.3%, with TOSCC23 cells showing the highest percentages of SP cells (3.3% of the total cell population). The SP cells isolated from TOSCC23 cells also showed greater cell proliferation and invasion compared to non-SP (MP) cells. Therefore, our initial findings suggested that SP cells were enriched for CSC-like cells. Furthermore, DNA microarray analysis revealed that the expression of cell proliferation-related and anti-apoptotic genes was greater in SP cells compared to MP cells. We focused on Lin28a, which showed the highest expression (approximately 22-fold) among the upregulated genes. The overexpression of Lin28a in TOSCC23 cells increased their proliferation, colony formation, and invasion. These findings suggest that Lin28a is an appropriate CSC target molecule for OSCC treatment.

Potential role of hematopoietic pre-B cell leukemia transcription factor-interacting protein in oral carcinogenesis.

BACKGROUND Hematopoietic pre-B-cell leukemia transcription factor-interacting protein (HPIP) is a corepressor of pre-B-cell leukemia homeobox (PBX) 1 and is known to play a role in hematopoiesis. Recently, HPIP was demonstrated to promote breast cancer cell proliferation and hepatocellular carcinoma growth. Moreover, it has been revealed that homeobox and PBX proteins, the expression of which is regulated by HPIP, play key roles in cancer of various organs, including oral squamous cell carcinoma (OSCC). Nevertheless, there has not been any study regarding the role of HPIP in OSCC. This study investigated the expression of HPIP in normal oral mucosa, epithelial precursor lesion (OEPL), and OSCC, and the functional roles of HPIP in OSCC cells and normal keratinocytes. MATERIALS AND METHODS Immunohistochemical analysis of HPIP, Ki-67, and involucrin was performed in OSCC specimens, and the change in involucrin expression following RNA interference treatment against HPIP was examined by quantitative RT-PCR and Western blot analysis in SCC9 and NHEK cells undergoing extracellular calcium-induced differentiation. Matrigel transwell and cell proliferation assays for both cell lines transfected with HPIP siRNA were also conducted. RESULTS HPIP expression increased in OEPL and OSCC specimens. In vitro analysis revealed that HPIP suppressed differentiation and proliferation of SCC9 cells and transwell migration of NHEK cells, while HPIP promoted invasion of SCC9 and proliferation of NHEK cells. However, HPIP has no significant effect on NHEK cell differentiation. CONCLUSION HPIP may play a critical role in oral carcinogenesis and is thus a potential target for anticancer therapy, with particular emphasis on its involvement in differentiation and migration/metastasis.

Cytology of low-grade cribriform cystadenocarcinoma in salivary glands: Cytological and immunohistochemical distinctions from other salivary gland neoplasms.

Low‐grade cribriform cystadenocarcinoma of the parotid gland is rare malignancy that is classified as a variant of cystadenocarcinoma. In routine cytologic slides from fine‐needle aspiration of a parotid gland, we found several pseudopapillary clusters comprising mucus‐producing cells. They included a few tumor cells having three‐dimensional nuclear atypia and slight hyperchromatism, although most of the tumor cells showed bland nuclei. Our initial cytological diagnosis was: “Indeterminate. Uncertain whether cystadenocarcinoma or cystadenoma.” The subsequent histological diagnosis was low‐grade cribriform cystadenocarcinoma. Immunohistochemical staining showed diffuse and strong reactivity for S‐100; tumor nests that were rimmed by p63+ cells, which suggests intraductal proliferation. Here, we report cytomorphological findings of this case, and discuss cytological and immunohistochemical distinctions between low‐grade cribriform cystadenocarcinoma and other salivary gland tumors, including a review of the literature. Diagn. Cytopathol. 2016;44:241–245.

Plasmablastic lymphoma of the maxillary sinus with intraoral manifestation caused by direct alveolar bone infiltration in an HIV‐negative patient

To the Editor: Plasmablastic lymphoma (PBL) of the oral cavity was first described by Delecluse et al. in 1997 as a new entity of a form of non-Hodgkin lymphoma (NHL) associated with human immunodeficiency virus (HIV) infection. They reported a series of sixteen highly malignant diffuse large B-cell lymphomas of the oral cavity with unique immunohistologic features. All their cases displayed morphologic features of diffuse large-cell lymphomas, but strikingly differed from them in that they showed minimal or absent expression of the leukocyte common antigen (CD45) as well as B-cell antigen CD20. Alternatively, the tumor cells showed constant expression of the characteristic plasma cell antigens (VS38c and CD38), frequent expression of CD79a, variable expression of cytoplasmic immunoglobulins and monoclonal rearrangement of the immunoglobulin heavy chain gene. PBL has been listed in the 2008 World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues as a non-Hodgkin B-cell lymphoma occurring predominantly in HIV-positive patients. PBLs account for approximately 2.6% of all HIV-related NHL. Here, we present a case of PBL of the maxillary sinus with intraoral manifestation caused by direct alveolar bone infiltration in an HIV-negative patient. A 50-year-old female had been treated with endodontologic management for spontaneous pain and increase of mobility of the left second molar of the maxilla for two months prior to tooth extraction, and thereafter, wound healing failed to occur. The patient was admitted to the Dental Hospital of Showa University for referral to oral surgery. On examination, a gingival ulcer, 15 × 12 mm in size, was found in the extraction site of the left second molar of the maxilla. Laboratory findings showed increases in lactate dehydrogenase (262 U/L), creatinine kinase (380 U/L) and γ-glutamic transferase (97 U/L), and revealed high thymol turbidity test (7.2 U). Complete blood count and differential peripheral white blood cell count were normal. All other laboratory data examined were within normal ranges. Computed tomography (CT) detected a lesion, 40 × 35 × 50 mm in size, occupying the left maxillary sinus, which partially destroyed the medial and posterior walls of the maxillary sinus, and extended into the nasal cavity with permeated pattern without definite central necrosis (Fig. 1a). Further, this lesion pressed against the left orbital floor, directly infiltrated into the alveolar bone of the left maxillary molar region, and caused intraoral manifestation (Fig. 1b). Nodal involvements were suggested in the left submandibular and cervical lymph nodes from diagnostic imaging. The patient had no history of autoimmune and lymphomatous diseases. There was no evidence of immunosuppression. Biopsy was performed from the gingival ulcer which showed that the lesion was composed of diffuse and cohesive monotonous proliferation of large atypical lymphoid cells with immunoblastic or plasmablastic features (Fig. 1c,d). The nuclei were usually oval with prominent nucleoli. Only a few plasmacytic-like differentiations were identified. Apoptotic cells and mitotic figures were frequently observed. A few tingible body macrophages were sporadically present. Immunohistochemically, the atypical lymphoid cells were positive for CD45 and partially positive for CD79a, but negative for CD20 (L26) (Fig. 2b) and PAX5. Regarding the expression of plasma cell-associated antigens in the atypical lymphoid cells, CD138 was sporadically positive and VS38c (Fig. 2a) was diffusely positive. These cells were also positive for MUM-1, and focally positive for EMA and CD30. Proliferation rate as assessed by Ki-67 staining was more than 90% (Fig. 2c). Definite immunoglobulin light chain restriction was not detected by assessment for immunoglobulin kappa and lambda light chain antibodies. In situ hybridization showed positivity for Epstein-Barr virusencoded RNA (EBER) (Fig. 2d). The atypical lymphoid cells were negative for Bcl2, Bcl6, Cyclin D1, CD3, CD5, UCHL-1, CD8, CD56, TIA-1, Granzyme B and HHV8 LNA. The diagnosis of PBL was made. The patient was referred to the Department of Hematology at another hospital for further examination and treatment. No other organ and bone marrow involvement was identified, and the patient was evaluated as Stage II (Ann Arbor staging system). Serological screening was negative for hepatitis B virus, hepatitis C virus and HIV. The patient was treated with four cycles of chemotherapy with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) and has been in complete remission. Recent examination of this patient at 9 months after chemotherapy showed no evidence of recurrence. To date, there have been four cases reported of PBL primarily arising in the maxillary sinus with HIV negativity, including the present case. Their ages ranged from 24 to 86 years, with a mean age of 51 years, and male-to-female ratio of 1:1. One patient died of disease at 4 months after diagnosis, while the other patients were all alive for more *Correspondence: Tarou Irié, e-mail: tarou@dent.showa-u.ac.jp Disclosure: No conflict of interest declared. †Contributed equally to this work. Pathology International 2014; 64: 588–590 doi:10.1111/pin.12212 bs_bs_banner