DOCK2 is involved in the host genetics and biology of severe COVID-19.

Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1-5. Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target.

The DNA integrity number and concentration are useful parameters for successful comprehensive genomic profiling test for cancer using formalin-fixed paraffin embedded tissue.

The acquisition of high-quality biospecimens and the appropriate handling of these materials are indispensable for successful clinical sequencing. We developed a cancer clinical sequencing system targeting 160 cancer genes: PleSSision-Rapid. Through the PleSSision-Rapid system, we have analyzed DNA quality evaluated by DIN (DNA integrity number) with 1329 formalin-fixed paraffin embedded (FFPE) samples including 477 prospectively collected tissues for genomic test (P) and 852 archival samples after routine pathological diagnosis (A1/A2). As a result, the samples with more than DIN 2.1 was 92.0% (439/477) in prospectively collected sample (P), while it was 85.6% (332/388) and 76.7% (356/464) in two types of archival samples (A1/A2). We performed the PleSSision-Rapid sequence using the samples with over DIN 2.1 and DNA concentration >10 ng/µL with which we were able to construct a DNA library, and the probability of sequence success was almost equivalent during all types of specimen processing, at 90.7% (398/439) in (P), 92.5% (307/332) in (A1) and 90.2% (321/356) in (A2), respectively. Our result indicated the clinical benefit to prepare the prospective collection of FFPE materials for indisputable clinical sequence, and that DIN ≥ 2.1 would be a solid parameter for sample preparation of comprehensive genomic profiling tests.

Clinical impact of a cancer genomic profiling test using an in-house comprehensive targeted sequencing system.

Precision medicine is a promising strategy for cancer treatment. In this study, we developed an in-house clinical sequencing system to perform a comprehensive cancer genomic profiling test as a clinical examination and analyzed the utility of this system. Genomic DNA was extracted from tumor tissues and peripheral blood cells collected from 161 patients with different stages and types of cancer. A comprehensive targeted amplicon exome sequencing for 160 cancer-related genes was performed using next-generation sequencing (NGS). The sequencing data were analyzed using an original bioinformatics pipeline, and multiple cancer-specific gene alterations were identified. The success rate of our test was 99% (160/161), while re-biopsy was required for 24% (39/161) of the cases. Potentially actionable and actionable gene alterations were detected in 91% (145/160) and 46% (73/160) of the patients, respectively. The actionable gene alterations were frequently detected in PIK3CA (9%), ERBB2 (8%), and EGFR (4%). High tumor mutation burden (TMB) (≥10 mut/Mb) was observed in 12% (19/160) of the patients. The secondary findings in germline variants considered to be associated with hereditary tumors were detected in 9% (15/160) of the patients. Seventeen patients (11%, 17/160) were treated with genotype-matched therapeutic agents, and the response rate was 47% (8/17). The median turnaround time for physicians was 20 days, and the median survival time after the initial visit was 8.7 months. The results of the present study prove the feasibility of implementing in-house clinical sequencing as a promising laboratory examination technique for precision cancer medicine.

Genomic testing for pancreatic cancer in clinical practice as real-world evidence.

BACKGROUND: Precision medicine guided by comprehensive genome sequencing represents a potential treatment strategy for pancreatic cancer. However, clinical sequencing for pancreatic cancer entails several practical difficulties. We have launched an in-house clinical sequencing system and started genomic testing for patients with cancer in clinical practice. We have analyzed the clinical utility of this system in pancreatic cancer. METHODS: We retrospectively reviewed 20 patients with pancreatic cancer who visited our division. Genomic DNA was extracted from both tumor tissue and peripheral blood mononuclear cells obtained from the patients. We performed a comprehensive genomic testing using targeted amplicon sequencing for 160 cancer-related genes. The primary endpoints were the detection rates of potential actionable and druggable gene alterations. The secondary endpoints were the detection rate of secondary germline findings, the rate of re-biopsy required for genome sequencing, survival time after the initial visit (post-sequencing survival time), and turnaround time. RESULTS: Although re-biopsy was required for 25% (5/20) of all patients, genomic testing was performed in all patients. Actionable and druggable gene alterations were detected in 100% (20/20) and 35% (7/20) of patients, respectively, whereas secondary germline findings were detected in 5% (1/20) of patients. The median turnaround times for physicians and patients were 20 and 26 days, respectively. The median post-sequencing survival time was 10.3 months. Only 10% (2/20) of all patients were treated with therapeutic agents based on the outcomes of genomic testing. CONCLUSIONS: The clinical application of comprehensive genomic testing for pancreatic cancer was feasible and promising in clinical practice.

Immunohistochemical expression profiles of solute carrier transporters in alpha-fetoprotein-producing gastric cancer.

AIMS: Alpha-fetoprotein (AFP)-producing gastric cancer (GC) is an aggressive tumour with high rates of liver metastasis and poor prognosis, and for which a validated chemotherapy regimen has not been established. Drug uptake by solute carrier (SLC) transporters is proposed as one of the mechanisms involved in sensitivity to chemotherapy. In this study, we aimed to develop important insights into effective chemotherapeutic regimens for AFP-producing GC. METHODS AND RESULTS: We evaluated immunohistochemically the expression levels of a panel of SLC transporters in 20 AFP-producing GCs and 130 conventional GCs. SLC transporters examined were human equilibrative nucleoside transporter 1 (hENT1), organic anion transporter 2 (OAT2), organic cation transporter (OCT) 2, OCT6 and organic anion-transporting polypeptide 1B3 (OATP1B3). The rates of high expression levels of hENT1 (hENT1high ) and OAT2 (OAT2high ) were statistically higher in AFP-producing GC, compared with conventional GC. When analysing hENT1 and OAT2 in combination, hENT1high /OAT2high was the most particular expression profile for AFP-producing GC, with a greater significance than hENT1 or OAT2 alone. However, no significant differences in OCT2, OCT6 or OATP1B3 levels were detected between AFP-producing and conventional GCs. However, immunoreactivity for hENT1, OAT2 and OCT6 tended to be increased in GC tissues compared with non-neoplastic epithelia. CONCLUSIONS: Because hENT1 and OAT2 are crucial for the uptake of gemcitabine and 5-fluorouracil, respectively, our results suggest that patients with AFP-producing GC could potentially benefit from gemcitabine/fluoropyrimidine combination chemotherapy. Increased expression of hENT1, OAT2 and OCT6 may also be associated with the progression of GC.

Triexponential function analysis of diffusion-weighted MRI for diagnosing prostate cancer.

BACKGROUND: To evaluate more detailed information noninvasively through on diffusion and perfusion in prostate cancer (PCa) using triexponential analysis of diffusion-weighted imaging (DWI). METHODS: Sixty-three prostate cancer patients underwent preoperative 3.0 Tesla MRI including eight b-values DWI. Triexponential analysis was performed to obtain three diffusion coefficients (Dp , Df , Ds ), as well as fractions (Fp , Ff , Fs ). Each diffusion parameter for cancerous lesions and normal tissues was compared and the relationship between diffusion parameters and Gleason score (GS) was assessed. K(trans) , Ve , and the ratios of intracellular components measured in histopathological specimens were compared with diffusion parameters. RESULTS: Dp was significantly greater for cancerous lesions than normal peripheral zone (PZ) (P < 0.001), whereas Dp in transition zone (TZ) showed no significant difference (P = 0.74, 95% confidence interval (CI) = -4.69-6.48). Ds was significantly smaller for each cancerous lesions in PZ and TZ (P < 0.001, respectively). There was no significant difference in Df between cancerous lesions and normal tissues in PZ and TZ (P = 0.07, 95% CI = -0.29-0.12 and P = 0.53, 95% CI = -3.51-2.29, respectively). D obtained with biexponential analysis were significantly smaller in cancerous lesions than in normal tissue in PZ and TZ (P < 0.001 for both), while D* in PZ and TZ showed no significant difference (P = 0.14, 95% CI = -1.60-0.24 and P = 0.31, 95% CI = -3.43-1.16, respectively). Dp in PZ and TZ showed significant correlation with K(trans) (R = 0.85, P < 0.001; R = 0.81, P < 0.001, respectively), while D(*) in PZ obtained with biexponential analysis showed no such correlation (P = 0.08, 95% CI = -0.14-0.30). Fs was significantly correlated with intracellular space fraction evaluated in histopathological specimens in PZ and TZ cancer (R = 0.41, P < 0.05; R = 0.59, P < 0.001, respectively). Ff and Fs correlated significantly with GS in PZ and TZ cancer (PZ: R = -0.44, P < 0.05; R = 0.37, P < 0.05, TZ: R = -0.59, P < 0.05; R = 0.57, P < 0.05, respectively). CONCLUSION: Triexponential analysis is a noninvasive approach that can provide more detailed information regarding diffusion and perfusion of PCa than biexponential analysis.

Stage-specific embryonic antigen-4 identifies human dental pulp stem cells.

Embryonic stem cell-associated antigens are expressed in a variety of adult stem cells as well as embryonic stem cells. In the present study, we investigated whether stage-specific embryonic antigen (SSEA)-4 can be used to isolate dental pulp (DP) stem cells. DP cells showed plastic adherence, specific surface antigen expression, and multipotent differentiation potential, similar to mesenchymal stem cells (MSC). SSEA-4+ cells were found in cultured DP cells in vitro as well as in DP tissue in vivo. Flow cytometric analysis demonstrated that 45.5% of the DP cells were SSEA-4+. When the DP cells were cultured in the presence of all-trans-retinoic acid, marked downregulation of SSEA-3 and SSEA-4 and the upregulation of SSEA-1 were observed. SSEA-4+ DP cells showed a greater telomere length and a higher growth rate compared to ungated and SSEA-4- cells. A clonal assay demonstrated that 65.5% of the SSEA-4+ DP cells had osteogenic potential, and the SSEA-4+ clonal DP cells showed multilineage differentiation potential toward osteoblasts, chondrocytes, and neurons in vitro. In addition, the SSEA-4+ DP cells had the capacity to form ectopic bone in vivo. Thus, our results suggest that SSEA-4 is a specific cell surface antigen that can be used to identify DP stem cells.

Evaluation of the usability of various rapid antibody tests in the diagnostic application for COVID-19.

BACKGROUND: The usability of laboratory tests related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critically important for the world undergoing the COVID-19 pandemic. The present study aimed to assess the diagnostic usability of rapid tests for the detection of antibody against SARS-CoV-2 through comparison of their results with the results of reverse transcription polymerase chain reaction (RT-PCR) test for the detection of SARS-CoV-2 genomic RNA and with the results of a quantitative test for antibody detection. METHODS: Serum samples were collected from 18 patients undergoing RT-PCR testing for SARS-CoV-2. Twelve patients were RT-PCR positive while six were negative. A quantitative test based on chemiluminescent immunoassay and three rapid tests based on immunochromatography were performed to detect anti-SARS-CoV-2 IgG and IgM. RESULTS: All the antibody tests exhibited poor sensitivity at the timing of initial RT-PCR diagnosis. IgG responses occurring prior to or simultaneously with IgM responses were observed through not only the quantitative test but also the three rapid tests. Based on concordance with the quantitative test results, the large variance among the three rapid tests was revealed. CONCLUSIONS: All antibody tests were unsatisfactory to replace RT-PCR for the early diagnosis of COVID-19. Rapid antibody tests as well as a quantitative antibody test were useful in the assessment of immune responses in COVID-19. The obvious variance among the three rapid tests suggested limited accuracy and difficult standardization. Diagnostic usability of rapid antibody tests for COVID-19 should be investigated rigorously.

Xenografts Derived From Patients' Ascites Recapitulate the Gemcitabine Resistance Observed in Pancreatic Cancer Patients.

OBJECTIVES: Most patient-derived pancreatic ductal adenocarcinoma (PDAC) xenografts have been established from surgical specimens of patients who have not received chemotherapy. However, xenografts have rarely been established from chemotherapy-resistant, advanced PDACs, because such cases are usually inoperable. The purpose of this study is to establish patient-derived xenografts using PDAC cells refractory to chemotherapy. METHODS: Clinical PDAC cells obtained from ascites of patients who had received continuous chemotherapy were implanted into the flanks of immunocompromised mice. Growth and histological features of the xenografts with and without gemcitabine treatment were then analyzed. RESULTS: Ascites-derived PDAC cells were successfully expanded through serial xenograft passage without changes in histological appearance. While treatment with gemcitabine substantially inhibited the growth of all PDAC xenografts tested, the tumor volume gradually increased, and the tumors showed marked regrowth even under continued gemcitabine treatment. These findings are consistent with the actual clinical course of the corresponding patients for each xenograft. CONCLUSIONS: Ascites-derived xenograft models represent a valuable experimental system for testing the efficacy of currently available therapeutic compounds on chemotherapy-resistant PDAC cells and for elucidation of the mechanisms underlying chemotherapy resistance.

Engineering cancer stem-like cells from normal human lung epithelial cells.

It has been proposed that a subpopulation of tumour cells with stem cell-like characteristics, known as cancer stem cells (CSCs), drives tumour initiation and generates tumour heterogeneity, thus leading to cancer metastasis, recurrence, and drug resistance. Although there has been substantial progress in CSC research into many solid tumour types, an understanding of the biology of CSCs in lung cancer remains elusive, mainly because of their heterogeneous origins and high plasticity. Here, we demonstrate that engineered lung cancer cells derived from normal human airway basal epithelial cells possessed CSC-like characteristics in terms of multilineage differentiation potential and strong tumour-initiating ability. Moreover, we established an in vitro 3D culture system that allowed the in vivo differentiation process of the CSC-like cells to be recapitulated. This engineered CSC model provides valuable opportunities for studying the biology of CSCs and for exploring and evaluating novel therapeutic approaches and targets in lung CSCs.

Psf3 is a prognostic biomarker in lung adenocarcinoma: a larger trial using tissue microarrays of 864 consecutive resections.

OBJECTIVES: Partner of Sld five (Psf) 3 is a member of the evolutionarily conserved heterotetrameric complex GINS (Go-Ichi-Ni-San). We previously reported that Psf3 could serve as a biomarker of poor prognosis in lung adenocarcinoma. Here, we used tissue microarrays to analyse Psf3 expression in lung adenocarcinoma and investigated whether its expression is associated with survival outcomes. METHODS: The study included 864 consecutive patients with lung adenocarcinoma who underwent complete resection at Hyogo Cancer Center between January 2002 and December 2009. Tissue microarrays were prepared, and Psf3 was detected using mouse antihuman Psf3 primary monoclonal antibodies. The status of Psf3 expression was determined using these microarrays. RESULTS: Of the 864 patients, 375 had high-positive Psf3 expression and 489 had low-positive expression. Psf3 expression was significantly associated with age, sex, T factor, lymph node metastasis, stage and P factor. The 5-year disease-free survival (DFS) rate was significantly lower in patients with high-positive Psf3 expression than in those with low-positive expression, and Psf3 expression, sex, age, T factor and lymph node metastasis were identified as independent and significant prognostic determinants. Among patients with Stage I adenocarcinoma, the 5-year DFS rate was significantly lower in those with high-positive Psf3 expression than in those with low-positive expression, and Psf3 expression was the most powerful survival predictor. CONCLUSIONS: The present findings strengthened our previous data demonstrating that high Psf3 expression in primary lung adenocarcinoma plays an important role in disease progression and is a prognostic indicator, particularly in early-stage adenocarcinoma.

Cell cycle kinetic analysis of colorectal neoplasms using a new automated immunohistochemistry-based cell cycle detection method.

We have recently developed a new method called the immunohistochemistry-based cell cycle detection (iCCD), which allows the determination of cell cycle phases on a cell-by-cell basis. This automated procedure can be performed on tissue sections and involves triple immunostaining for geminin, cdt1, and γ H2A.X, which are nuclear proteins expressed sequentially, with a few overlaps, during the cell cycle. In the current study, we applied this technique to resected specimens of colorectal neoplasm to determine the usefulness of iCCD for the pathological examination of colorectal cancers. We examined 141 cases of colorectal cancers. Normal mucosa and adenomas were analyzed as controls. In nonneoplastic mucosa, we observed a pattern of distribution of the cells positive for these cell cycle markers. Adenomas showed a slight distortion in this pattern, the geminin-positive cells, indicative of S/G2/M phase, were localized in the upper one-third region of the crypts. In neoplastic mucosa, the marker expression pattern was disorganized. Compared with normal mucosa, colorectal neoplasms showed an increased proportion of geminin-positive cells and decreased percentages of cdt1-positive cells (G1 phase). However, we did not find significant difference in the expression pattern between adenomas and carcinomas. Cellular proportions were correlated with clinicopathological parameters such as microscopic vascular invasion and pT stages. In cases of preoperative adjuvant therapy, the proportion of geminin-positive cells decreased, whereas that of γ H2A.X-positive cells (indicative of apoptosis/degeneration) increased significantly. We believe that this novel method can be applied to clinical samples to evaluate cell cycle kinetics and the effects of preoperative adjuvant therapy in colorectal cancers.

Immunohistochemical profiling of ALK fusion gene-positive adenocarcinomas of the lung.

Our aim was to determine whether or not non-small-cell lung cancer is squamous cell carcinoma (SQCC); even in small samples, it is essential in view of the side effects attendant on new therapeutics. Lung adenocarcinoma (ADC) with the EML4-ALK fusion gene has been described as demonstrating mucinous cribriform/acinar growth and signet-ring cells, sometimes partially simulating SQCC. We investigated the relation among morphology, anaplastic lymphoma kinase (ALK) rearrangement, and immunophenotype in 321 ADCs by tissue microarray using SQCC markers cytokeratin (CK)5/6, CK14, desmocollin-3, desmoglein-3, p40, p63 versus ADC markers thyroid transcription factor (TTF)-1 and napsin A. Unlike 312 ALK-negative ADCs, 9 ALK-positive cases were negative for 4 SQCC markers. Only 1 ALK-positive ADC showing assertive morphology was positive for CK5/6 and p63 as well as for TTF-1 and napsin A. Coexpression of TTF-1/p40 was not observed, unlike that of TTF-1/p63 reported previously. There was no statistically significant difference between ALK-negative and ALK-positive ADC by immunohistochemical profiling.

Immunohistochemistry-based cell cycle detection (iCCD): a novel system to visualize cell kinetics on formalin-fixed paraffin-embedded tissues.

Carcinogenesis is widely believed to occur when regulatory systems governing cellular proliferation and differentiation are compromised. To date, various methods have been devised to determine cell cycle. However, these methods have not gained popularity in the diagnostic field. We developed a multiplex immunohistochemical method that can simultaneously stain cells in the G1 and S/G2/M phases and those undergoing apoptosis with the 3 markers Cdt1, geminin, and gamma H2A.X. The staining procedure can be performed using an autoimmunostainer. The nuclei of cells in the G1 phase stain red with the antibody for Cdt1, those in the S/G2/M phases stain blue with the antibody for geminin, and the nuclei of cells undergoing apoptosis stain brown with the antibody for H2A.X. The present method enables accurate cell cycle assessments using paraffin-embedded tissue specimens, which are superior to other forms of specimens in terms of morphologic observation.

Rapid multiplex immunohistochemistry using the 4-antibody cocktail YANA-4 in differentiating primary adenocarcinoma from squamous cell carcinoma of the lung.

The current Food and Drug Administration-approved standard treatment for non-small cell carcinomas consists of carboplatin/taxol/avastin. However, nowadays more specialized protocols, depending on tumor subtype, are being used for lung cancer patients. Therefore, accurate differentiation between adenocarcinoma and squamous cell carcinoma is essential for the selection of appropriate therapies. We designed a rapid multiplex immunostaining method using a novel 4-antibody cocktail, YANA-4. This antibody cocktail consists of the following monoclonal antibodies: rabbit for thyroid transcription factor 1(TTF-1), mouse for napsin A, mouse l for p63, and rabbit for CK14. All procedures can be completed within 3 hours. This method labels the nuclei of adenocarcinomas as brown with TTF-1, and cytoplasm as blue with napsin A. Squamous cell carcinomas could be differentiated from adenocarcinomas with an inverse staining pattern: blue nuclei with p63 and brown cytoplasm with CK14. In this study, 97.4% (38 of 39) of adenocarcinomas showed brown nuclei (TTF-1) and/or blue cytoplasm (napsin A), with 4 cases showing positivity only for brown nuclei (TTF-1) and 1 case only for blue cytoplasm (napsin A). None of the squamous cell carcinoma cases showed these staining patterns. Positivity for blue nuclei (p63) and/or brown cytoplasm (CK14) was detected in 100% (25 of 25) of squamous cell carcinomas, with 1 case showing positivity only for brown cytoplasm (CK14) and 2 cases only for blue nuclei (p63). None of the adenocarcinoma cases showed these patterns. This rapid immunohistochemical method can thus be considered highly specific and sensitive for differentiating adenocarcinomas and squamous cell carcinomas.

Oncogene-mediated human lung epithelial cell transformation produces adenocarcinoma phenotypes in vivo.

It has been challenging to engineer lung adenocarcinoma models via oncogene-mediated transformation of primary cultured normal human cells. Although viral oncoprotein-mediated malignant transformation has been reported, xenografts derived from such transformed cells generally represent poorly differentiated cancers. Here, we demonstrate that the combined expression of multiple cellular factors induces malignant transformation in normal human lung epithelial cells. Although a combination of four genetic alterations, including hTERT overexpression, inactivation of the pRB and p53 pathways, and KRAS activation, is insufficient for normal human small airway epithelial cells to be fully transformed, expression of one additional oncogene induces malignant transformation. Notably, we have succeeded in reproducing human lung adenocarcinoma phenotypes in the flanks of nude mice by introducing an active form of PIK3CA, CYCLIN-D1, or a dominant-negative form of LKB1 in combination with the four genetic alterations above. Besides differentiated lung cancer, poorly differentiated cancer models can also be engineered by employing c-MYC as one of the genetic elements, indicating that histologic features and degree of differentiation of xenografts are controllable to some extent by changing the combination of genetic elements introduced. This is the first study reporting malignant transformation of normal lung epithelial cells in the absence of viral oncoproteins. We propose that our model system would be useful to identify the minimal and most crucial set of changes required for lung tumorigenesis, and that it would provide a broadly applicable approach for discovering attractive therapeutic targets.

Preventive effect of chemical peeling on ultraviolet induced skin tumor formation.

BACKGROUND: Chemical peeling is one of the dermatological treatments available for certain cutaneous diseases and conditions or improvement of cosmetic appearance of photoaged skin. OBJECTIVES: We assessed the photochemopreventive effect of several clinically used chemical peeling agents on the ultraviolet (UV)-irradiated skin of hairless mice. METHODS: Chemical peeling was done using 35% glycolic acid dissolved in distilled water, 30% salicylic acid in ethanol, 10% or 35% trichloroacetic acid (TCA) in distilled water at the right back of UV-irradiated hairless mice every 2 weeks in case of glycolic acid, salicylic acid, and 10% TCA and every 4 weeks in case of 35% TCA for totally 18 weeks after the establishment of photoaged mice by irradiation with UVA+B range light three times a week for 10 weeks at a total dose of 420 J/cm(2) at UVA and 9.6 J/cm(2) at UVB. Tumor formation was assessed every week. Skin specimens were taken from treated and non-treated area for evaluation under microscopy, evaluation of P53 expression, and mRNA expression of cyclooxygenase (COX)-2. Serum level of prostaglandin E(2) was also evaluated. RESULTS: All types of chemical peeling reduced tumor formation in treated mice, mostly in the treated area but also non-treated area. Peeling suppressed clonal retention of p53 positive abnormal cells and reduced mRNA expression of COX-2 in treated skin. Further, serum prostaglandin E(2) level was decreased in chemical peeling treated mice. CONCLUSIONS: These results indicate that chemical peeling with glycolic acid, salicylic acid, and TCA could serve tumor prevention by removing photodamaged cells.

Pharmacogenomics of metabotropic glutamate receptor subtype 1 and in vivo malignant melanoma formation.

We have previously shown that ectopic expression of metabotropic glutamate receptor subtype 1 in melanocytes is essential for both development and in vivo growth of melanoma using newly developed transgenic mice which conditionally express metabotropic glutamate receptor subtype 1 (mGluR1). In this study, we developed conditional transgenic mice, which harbor melanocytes not only in the dermis and hair follicles but also in the epidermis using stem cell factor transgenic mice. Pigmented plaques on the backs, tails, ears or groins of the transgenic mice began to appear 13 weeks after activation of the mGluR1 transgene, and the transgenic mice produced melanomas at a frequency of 100% 36 weeks after transgene activation. Although this transgenic mouse harbors melanocytes in the epidermis, proliferation of melanoma cells took place in the dermis. To elucidate the signals involved in development and growth of melanoma, inhibitors to phospholipase C, protein kinase C and mitogen-activated protein kinase kinase 1/2, and antagonists to Ca(2+) and calmodulin were administrated to transgenic mice. Each signal inhibitor to phospholipase, protein kinase C, Ca(2+) release, calmodulin and mitogen-activated protein kinase kinase 1/2 inhibited melanoma development. However, once melanoma was developed, the growth of melanoma was dramatically inhibited only by the inhibitor to mitogen-activated protein kinase kinase 1/2 with partial inhibition by inhibitors to protein kinase C and phospholipase C. This inhibition of melanoma growth was well correlated with the expression of phosphorylated extracellular signal-regulated kinase 1/2 and Ki-67. These results indicate that for development of melanoma, activation of every signaling pathway from mGluR1 is required. However, for growth of melanoma, the extracellular signal-regulated kinase pathway plays a key role.

Effect of chemical peeling on photocarcinogenesis.

Chemical peeling is one of the dermatological treatments available for certain cutaneous diseases and conditions or improvement of cosmetic appearance of photo-aged skin. We assessed the photo-chemopreventive effect of several clinically used chemical peeling agents on the ultraviolet-irradiated skin of hairless mice. Chemical peeling was done using 35% glycolic acid dissolved in distilled water, 30% salicylic acid in ethanol, and 10% or 35% trichloroacetic acid in distilled water at the right back of ultraviolet-irradiated hairless mice every 2 weeks for glycolic acid, salicylic acid and 10% trichloroacetic acid, and every 4 weeks for 35% trichloroacetic acid for a total of 18 weeks after the establishment of photo-aged mice by irradiation with ultraviolet B range light three times a week for 14 weeks at a total dose of 6.66 J/cm(2) . Tumor formation was assessed every week. Skin specimens were taken from treated and non-treated area for evaluation under microscopy, evaluation of p53 expression and mRNA expression of cyclooxygenase-2. Serum level of prostaglandin E(2) was also evaluated. All types of chemical peeling reduced tumor formation in treated mice, mostly in the treated area but also in the non-treated area. Peeling suppressed retention of p53-positive abnormal cells and reduced mRNA expression of cyclooxygenase-2 in treated skin. Further, serum prostaglandin E(2) level was decreased in chemical peeling treated mice. These results indicate that chemical peeling with glycolic acid, salicylic acid and trichloroacetic acid could serve tumor prevention by removing photo-damaged cells.