Tsuyoshi Asato

A Large Case-Control Study of Cervical Cancer Risk Associated with Human Papillomavirus Infection in Japan, by Nucleotide Sequencing-Based Genotyping

Using nucleotide sequencing-based genotyping, we conducted a case-control study to examine cervical cancer risk associated with human papillomavirus (HPV) infection in a Japanese population. A consensus primer pair was used to amplify DNA from the L1 region of HPV by polymerase chain reaction (PCR). By PCR, 311 of 356 patients with cervical cancer and 333 of 3249 control individuals were positive for HPV. By the direct sequencing of PCR products, nucleotide sequences of 30 genotypes were obtained. A high incidence of type 52 and a low incidence of type 16 were characteristic of the control group. Odds ratios were estimated for 18 genotypes. Types 71, 90, and 91, previously uncharacterized, were classified as low-risk genotypes, which is consistent with predictions made on the basis of phylogeny. The present study is the first large case-control study of its kind to use nucleotide sequencing-based genotyping.

Detection of human papillomavirus DNA in primary and metastatic lesions of carcinoma of the cervix in women from Okinawa, Japan.

Of 351 patients with invasive cervical cancer treated at Ryukyu University Hospital, Okinawa, 293 who were tested for human papilloma virus (HPV) DNA of the primary cervical lesion before the initiation of treatment were considered for the study. The polymerase chain reaction (PCR) was performed using fresh specimens, immediately after sampling. In 250 of 293 patients (85.3%), HPV DNA was detected in cervical tumor by PCR using L1 consensus primer. The positive rate by histology was 89.9% in squamous cell carcinoma, 93.8% in adenosquamous carcinoma, and 51.4% in adenocarcinoma. The former two figures were significantly higher than the latter (p < 0.001 and p = 0.002). Concerning identification of HPV types, HPV 16 was most predominant in squamous cell carcinoma, whereas type 18 was relatively high in adenocarcinoma. However, the type distribution of HPV was different to some extent from those in other countries. During treatment, 489 nodal and other tissue samples were obtained from 113 of 250 HPV DNA–positive patients, and were submitted to an assay of HPV DNA. HPV DNA was amplified in all 55 metastatic samples and also in 12 of 434 nonmetastatic tissues (2.8%). HPV types specified in these samples were always identical with the HPV types determined in their primary tumors. In 154 samples from 29 of 43 HPV DNA–negative patients, HPV DNA was not detected, either in 14 metastatic samples or in 140 histologically benign samples. Cancer-free, but HPV DNA–positive nodal, liver, and pulmonary tissues could be interpreted to be already involved at the time of examination, by observing the clinical course of the disease over time.

MINK is a Rap2 effector for phosphorylation of the postsynaptic scaffold protein TANC1.

Rap1 and Rap2 are similar Ras-like G proteins but perform distinct functions. By the affinity chromatography/mass-spectrometry approach and the yeast two-hybrid screening, we identified Misshapen/NIKs-related kinase (MINK) as a novel Rap2-interacting protein that does not interact with Rap1 or Ras. MINK is a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases. The interaction between MINK and Rap2 was GTP-dependent and required Phe39 within the effector region of Rap2; the corresponding residue in Rap1 and Ras is Ser. MINK was enriched in the brain, and both MINK and its close relative, Traf2- and Nck-interacting kinase (TNIK), interacted with a postsynaptic scaffold protein containing tetratricopeptide repeats, ankyrin repeats and a coiled-coil region (TANC1) and induced its phosphorylation, under control of Rap2 in cultured cells. These are novel actions of MINK and TNIK, and consistent with a role of MINK as a Rap2 effector in the brain.

Alterations of protein kinase C, 8-hydroxydeoxyguanosine, and K-ras oncogene in rat lungs exposed to passive smoking

To investigate the effects of exposure to sidestream cigarette smoke (CS) on the initiation and promotion of lung cancer, two groups of 8 or 10 rats were exposed to CS for a 1 h period twice a day for 8, 12, or 20 weeks. The protein kinase C (PKC) activity of the lung exhibited significant changes of 120, 86 and 81% in the CS groups, compared with the respective control group values in the three exposure periods. The in vitro activation of PKC by the active oxygens was efficiently eliminated by hydroxyl radical scavengers, indicating that hydroxyl radicals are responsible for the PKC activation. For the alterations in the lung nucleus caused by passive smoking, the 12- and 20-week exposure CS groups showed significant increases in the accumulation of 8-hydroxydeoxyguanosine. One rat with K-ras activation by G:C transversion (GGT-->GCT) at codon 12 was found among 26 rats of the CS groups in the three exposure periods. These results show that active oxygens introduced by passive smoking may contribute to K-ras activation as an initiator of a tumor model, possibly through the oxygen-induced DNA damage, and may also contribute to an initial activation and the subsequent down-regulation of PKC as a promoter.

Ectopic coexpression of keratin 8 and 18 promotes invasion of transformed keratinocytes and is induced in patients with cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) results from transformation of epidermal keratinocytes. Invasion of transformed keratinocytes through the basement membrane into the dermis results in invasive cSCC with substantial metastatic potential. To better understand the mechanisms for invasion and metastasis, we compared the protein expression profiles of a non-metastatic transformed mouse keratinocyte line and its metastatic derivative. Keratin 8 (Krt8) and Krt18, not seen in normal keratinocytes, were coexpressed and formed Krt8/18 filaments in the metastatic line. The metastatic line efficiently invaded an artificial basement membrane in vitro owing to the Krt8/18-coexpression, since coexpression of exogenous Krt8/18 in the non-invasive parental line conferred invasiveness. To test whether the Krt8/18-coexpression is induced and is involved in cSCC invasion, we examined specimens from 21 pre-invasive and 24 invasive cSCC patients by immunohistochemistry, and the ectopic Krt8/18-coexpression was almost exclusively found in invasive cSCC. Further studies are needed to examine the clinical significance of ectopic Krt8/18-coexpression in cSCC.

Rap2 function requires palmitoylation and recycling endosome localization

Rap2A, Rap2B, and Rap2C are Ras-like small G proteins. The role of their post-translational processing has not been investigated due to the lack of information on their downstream signaling. We have recently identified the Traf2- and Nck-interacting kinase (TNIK), a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases, as a specific Rap2 effector. Here we report that, in HEK293T cells, Rap2A (farnesylated) and Rap2C (likely farnesylated), but not Rap2B (geranylgeranylated), require palmitoylation for membrane-association and TNIK activation, whereas all Rap2 proteins, including Rap2B, require palmitoylation for induction of TNIK-mediated phenotype, the suppression of cell spreading. Furthermore, we report for the first time that, in COS-1 cells, Rap2 proteins localize, and recruit TNIK, to the recycling endosomes, but not the Golgi nor the endoplasmic reticulum, in a palmitoylation-dependent manner. These observations implicate the involvement of palmitoylation and recycling endosome localization in cellular functions of Rap2 proteins.

Association of human papillomavirus type 16 with malignant melanoma

We report a case of malignant melanoma associated with human papillomavirus (HPV) in a 37-year-old woman. The patient has had numerous brown papular and nodular tumors, 5 to 30 mm in diameter, on her left leg for > 15 years, some of them coalescing rapidly in the last 12 months to a multilobulated black nodule diagnosed as malignant melanoma by histology and immunohistochemistry. HPV type 16 DNA was detected in the melanoma specimen by reverse transcriptase polymerase chain reaction (rt-PCR) and in situ hybridization (ISH) of the tumor tissues. This is the first report of melanoma associated with HPV 16.

Detection of human papilloma virus type 56 in extragenital Bowen's disease

A case of Bowens disease arising on the medial part of the first metatarsal bone of an 81-year-old Japanese woman is described. Histopathologically, proliferation of atypical cells was found throughout the epidermis. Electronmicroscopy revealed virus particles 40-50 nm in diameter in the nuclei of tumour cells at the granular cells just on or below the horny layer. Positive bands were obtained by polymerase chain reaction using a consensus primer of human papilloma virus L1 portion. Sequencing analysis of the amplified DNA revealed the same base sequences and homology as human papilloma virus 56. To the best of our knowledge, this case is the first report in which human papilloma virus 56 was found in a case of extragenital Bowens disease. We consider it important to understand that human papilloma virus 56, often found in cervical lesions, can be detected in extragenital Bowens diseases.

Prevalence of HPV infection in cervical cytology‐normal women in Okinawa, Japan, as determined by a polymerase chain reaction

Epidemiological studies have shown that onset of sexual activity at early age and sexual promis- cuity are risk factors for the development of cer- vical cancer, possibly related to human papillo- . wx mavirus HPV infection 1 . In this context, studying the prevalence of HPV in a population with high incidence rate of cervical cancer can be informative for investigating the etiology of the disease. We analyzed the age-related prevalence of HPV in a large number of Okinawan women with cytologically normal cervices, using the po- . lymerase chain reaction PCR . In the Okinawa prefecture which is the southern-most part of Japan in the subtropical zone, the incidence rate of cervical cancer is reported to be the highest in

Angiopoietin-like protein 2 induces proinflammatory responses in peritoneal cells.

Monocytes and macrophages are important effectors and regulators of inflammation, and both their differentiation and activation are regulated strictly in response to environmental cues. Angiopoietin-like protein 2 (Angptl2) is a multifaceted protein, displaying many physiological and pathological functions in inflammation, angiogenesis, hematopoiesis, and tumor development. Although recent studies implicate Angptl2 in chronic inflammation, the mechanisms of inflammation caused by Angptl2 remain unclear. The purpose of the present study was to elucidate the role of Angptl2 in inflammation by understanding the effects of Angptl2 on monocytes/macrophages. We showed that Angptl2 directly activates resident murine peritoneal monocytes and macrophages and induces a drastic upregulation of the transcription of several inflammatory genes including nitric oxide synthase 2 and prostaglandin-endoperoxide synthase 2, and several proinflammatory cytokine genes such as interleukin (IL)-1β, IL-6, TNFα, and CSF2, along with activation of ERK, JNK, p38, and nuclear factor kappa B signaling pathways. Concordantly, proinflammatory cytokines IL-1β, IL-6, TNFα, and GM-CSF, were rapidly elevated from murine peritoneal monocytes and macrophages. These results demonstrate a novel role for Angptl2 in inflammation via the direct activation of peritoneal monocytes and macrophages.