p27 deregulation by Skp2 overexpression induced by the JAK2V617 mutation.

Janus kinase 2 (JAK2) V617F mutation has been regarded as the major cause of myeloproliferative disorders (MPD). However, the mechanisms of abnormal cell growth by JAK2V617F have not been elucidated. In this study, cell cycle regulatory protein expression was analyzed using JAK2V617F-Ba/F3 and mock-Ba/F3. JAK2V617F-Ba/F3, but not mock-Ba/F3, showed IL-3 independent cell growth and constitutive STATs activation. Deregulation of p27(Kip1), the cell cycle regulator at the G1 to S transition, was observed in JAK2V617F-Ba/F3 but not in mock-control. p27(Kip1) deregulation was not due to p27(Kip1) mRNA level but due to high Skp2 expression, a subunit of ubiquitin E3 ligase, through the STAT binding in the Skp2 promoter. Like JAK2V617F overexpression, constitutively active STAT5 or STAT3 induced aberrant p27(Kip1) expression of Ba/F3 cells. Similar findings were observed in BCR/ABL-transfected Ba/F3. Our results elucidate the regulatory mechanism by which JAK2V617F modulates Skp2 gene expression through the STAT transcription factors.

GATA-1 and GATA-2 binding to 3' enhancer of WT1 gene is essential for its transcription in acute leukemia and solid tumor cell lines.

Although oncogenic functions and the clinical significance of Wilms tumor 1 (WT1) have been extensively studied in acute leukemia, the regulatory mechanism of its transcription still remains to be determined. We found a significant correlation among the amounts of WT1, GATA-1 and GATA-2 mRNAs from leukemia and solid tumor cell lines. Overexpression and small interfering RNA (siRNA) transfection experiments of GATA-1 and GATA-2 showed that these GATA transcription factors could induce WT1 expression. Promoter analysis showed that the 5' promoter did not explain the different WT1 mRNA levels between cell lines. The 3' enhancer, especially the distal sites out of six putative GATA binding sites located within the region, but not the intron 3 enhancer, were essential for the WT1 mRNA level. Electrophoretic mobility shift assay (EMSA) showed both GATA-1 and GATA-2 bound to these GATA sites. Besides acute leukemia cell lines, solid tumor cell lines including, TYK-nu-cPr also showed a high level of WT1 mRNA. We showed that GATA-2 expression is a determinant of WT1 mRNA expression in both TYK-nu-cPr cells and HL60 cells without GATA-1 expression. Taken together, these results suggest that GATA-1 and/or GATA-2 binding to a GATA site of the 3' enhancer of WT1 played an important role in WT1 gene expression.

v-Src oncogene product increases sphingosine kinase 1 expression through mRNA stabilization: alteration of AU-rich element-binding proteins.

Sphingosine kinase 1 (SPHK1) is overexpressed in solid tumors and leukemia. However, the mechanism of SPHK1 overexpression by oncogenes has not been defined. We found that v-Src-transformed NIH3T3 cells showed a high SPHK1 mRNA, SPHK1 protein and SPHK enzyme activity. siRNA of SPHK1 inhibited the growth of v-Src-NIH3T3, suggesting the involvement of SPHK1 in v-Src-induced oncogenesis. v-Src-NIH3T3 showed activations of protein kinase C-alpha, signal transducers and activators of transcription 3 and c-Jun NH(2)-terminal kinase. Their inhibition suppressed SPHK1 expression in v-Src-NIH3T3, whereas their overexpression increased SPHK1 mRNA in NIH3T3. Unexpectedly, the nuclear run-on assay and the promoter analysis using 5'-promoter region of mouse SPHK1 did not show any significant difference between mock- and v-Src-NIH3T3. Furthermore, the half-life of SPHK1 mRNA in mock-NIH3T3 was nearly 15 min, whereas that of v-Src-NIH3T3 was much longer. Examination of two AU-rich region-binding proteins, AUF1 and HuR, that regulate mRNA decay reciprocally, showed decreased total AUF1 protein associated with increased tyrosine-phosphorylated form and increased serine-phosphorylated HuR protein in v-Src-NIH3T3. Modulation of AUF1 and HuR by their overexpression or siRNA revealed that SPHK1 mRNA in v-Src- and mock-NIH3T3 was regulated reciprocally by these factors. Our results showed, for the first time, a novel mechanism of v-Src-induced SPHK1 overexpression.

Implications of sphingosine kinase 1 expression level for the cellular sphingolipid rheostat: relevance as a marker for daunorubicin sensitivity of leukemia cells.

We recently reported increased sphingosine kinase 1 (SPHK1) and decreased neutral sphingomyelinase 2 (NSMase2) gene expression in myelodysplastic syndromes and acute leukemia. This alteration is supposed to change the cellular sphingolipid metabolites; however, positive correlations were observed between daunorubicin (DA)-IC50 and the SPHK1 message but not between DA-IC50 and NSMase2 messages, when 16 different leukemia cell lines were used to analyze the relationship between gene expressions and chemosensitivity against DA. Using two cell lines with either the highest or lowest SPHK1 expression, cellular ceramides and sphingosine 1-phosphate (S1P) were quantified by liquid chromatography/mass spectrometry. Increased ceramide was observed in DA-sensitive, but not in DA-resistant cell lines treated with low doses of DA. Upon DA treatment, S1P decreased more in the sensitive cell lines than in resistant cell lines. A SPHK inhibitor recovered the DA sensitivity of DA-resistant cells. The modulation of SPHK1 gene expression by either overexpression or using siRNA affected the DA sensitivity of representative cell lines. Results clearly show that SPHK1 is both a good marker to predict the DA sensitivity of leukemia cells and a potential therapeutic target for leukemia with high SPHK1 expression, and suggest that the sphingolipid rheostat plays a significant role in DA-induced cytotoxicity.

Are stable postoperative biliary atresia patients really stable?

Transforming growth factor-beta 1 (TGF beta-1) is an important mediator of liver-cell proliferation and replication that is implicated in hepatic fibrosis (HF). Hepatic stellate cells (HSC) are activated by TGF beta-1 and are the main precursor cells involved in fibrogenesis. The correlation between serum TGF beta-1, activated HSC in liver-biopsy specimens, and liver biochemistry was investigated to determine the value of TGF beta-1 as an indicator of clinical status in postoperative biliary atresia (BA) patients. Thirty-two postoperative BA patients (mean age 11.2 +/- 2.8 years) and 13 normal controls (mean age 10.3 +/- 3.7 years) were studied. Based on average liver function test (LFT) results over a 3-month period immediately prior to this study, the BA patients were divided into group I (anicteric, normal LFT; n = 10); group II (anicteric, elevated liver transaminases; n = 12), and group III (jaundiced end-stage liver fibrosis awaiting liver transplantation; n = 10). Serum TGF beta-1 was determined using ELISA. Liver-biopsy specimens were examined with antibody against TGF beta-1 and alpha-smooth muscle actin (SMA) antibody for detection of activated HSC. Serum TGF beta-1 was significantly higher in groups I (11.4 +/- 3.7 ng/ml; P < 0.01) and II (23.3 +/- 11.3 ng/ml; P < 0.001) than in group III (3.0 +/- 1.5 ng/ml) and controls (4.5 +/- 2.5 ng/ml) despite normal LFT in group I. The 3 subjects with the highest serum TGF beta-1 in group II had bile lakes. Biopsies from groups I and II were strongly positive for TGF beta-1 in hepatocytes and Kupffer cells and for activated HSC detected by SMA compared with group III and controls. Because serum TGF beta-1 and activated HSC are only present during active fibrosis, we conclude that there is progressive fibrogenesis even in seemingly normal postoperative BA patients. In particular, bile lakes should be regarded as a key sign of progressive HF, the presence of which should be regarded with extreme caution. We suggest that serum TGF beta-1 could be used as an accurate indicator of progressive fibrogenesis in postoperative BA patients.

Decreased GlcNAc 6-O-sulfotransferase activity in the cornea with macular corneal dystrophy.

PURPOSE: Macular corneal dystrophy (MCD) is an autosomal recessive inherited disorder that is accompanied by corneal opacity. Explants from MCD-affected corneas have been reported to synthesize low-sulfated KS, suggesting that sulfate groups attached to KS may play critical roles in maintaining corneal transparency. To clear the biosynthetic defect in the MCD cornea, sulfotransferase activities were determined that are presumably involved in the biosynthesis of KS: galactose-6-sulfotransferase (Gal6ST) activity and N-acetylglucosamine 6-O-sulfotransferase (GlcNAc6ST) activity. METHODS: Gal6ST and GlcNAc6ST activities, which were contained in the corneal extracts from corneas affected by MCD and keratoconus and from normal control corneas, were determined by measuring the transfer of (35)SO(4) from [(35)S]3'-phosphoadenosine 5'-phosphosulfate into the Gal residue of partially desulfated KS and the nonreducing terminal GlcNAc residue of GlcNAcbeta1-3Galbeta1-4GlcNAc (oligo A), respectively. RESULTS: The level of Gal6ST activity in corneal extracts from eyes with MCD, which was measured by using partially desulfated KS as an acceptor, was nearly equal to that in eyes with keratoconus and normal control eyes. In contrast, GlcNAc6ST activity in the extracts from MCD-affected corneas, which was measured by using oligo A as an acceptor, was much lower than in those in corneas with keratoconus and in normal control corneas. CONCLUSIONS: The decrease in GlcNAc6ST activity in the cornea with MCD may result in the occurrence of low- or nonsulfated KS and thereby cause corneal opacity.

[A clinical study on 109 cases with testicular tumor].

One hundred and nine patients with testicular tumors were treated in our hospital between 1970 and 1994. The patients ranged in age from 4 months to 73 years with an average of 35.3 years. The most frequent chief complaint was painless swelling of the scrotal contents. Of 106 germinal tumors, 61 cases were of pure seminomas, and the other 45 cases contained non-seminomatous elements. The remaining 3 cases were non-germinal tumors; 2 malignant lymphomas and one rhabdomyosarcoma. Clinical staging classified them into 80 cases of stage I, 21 cases of stage II, and 8 cases of stage III. The five-year survival rate calculated by the Kaplan-Meier method was 79.6% in total, and 85.0, 91.7, 66.7 and 50.0% in stage I, IIA, IIB, and III, respectively. A comparison with the 5-year survival rate before 1980 (69.9%) when computed tomographic (CT) equipment and chemotherapy including cisplatin were introduced in our hospital, showed significant improvement after 1981 (89.4%).

[Studies on skin irritation with urostomy--the gross and histologic evaluation of the attached area with urostomy].

The degree of damage on the peristomal skin of 42 patients who accepted the urostomy was visually and histologically evaluated. The incidence of damages on the areas of the skin where the materials was applied was 85.7%. The occurrence of non-active lesions (mainly permanent pigment change) was statistically more significant than the active lesions (mainly redness) [p < 0.05]. The incidence of skin lesions using the skin barrier agents was significantly lower statistically than the incidence with the adhesive medication indicating a superior treatment for the skin condition [p < 0.05]. Although the gross appearances were normal, the microscopic and electron microscopic evaluations detected chronic damages indicating comparatively evident changes. The results suggest that these histological evaluations are useful in examining the damage on peristomal skin.

[Clinical study of the advanced testicular tumor non-achieved complete response by primary chemotherapy and relapse cases. The East Japan Testicular Tumor Study Group].

The East Japan Testicular Tumor Study group has studied advanced testicular tumors. Treatment and prognosis of the patients who did not achieved complete response on the primary chemotherapy were discussed in this paper. Also 8 patients who had once reached complete response by primary treatments and relapsed later have been clinically analyzed. 1. Concerning the patients non-achieving complete response on the primary chemotherapy: Two cases who had resection of the residual tumors and 4 cases who had radiation therapy had reached NED in 7 cases of seminoma stage II. Nine of 10 cases in non-seminoma stage II reached NED on resection of the residuals. Moreover, 10 out of 17 cases in stage III showed NED on excision of the residuals. On the other hand, recuperating was very difficult in the cases who could not have excision of residual tumor. The histological findings of the residual tumors after chemotherapy were necrosis and fibrosis in 13 cases, viable cells in 4 cases, teratoma in 6 cases and other 2 cases were not clear. Five out of 15 who had teratoma in the primary lesion had teratoma in the resected residuals on metastatic lesion. On the other hand 5 patients who did not have teratoma originally had no any teratoma elements in the residuals. 2. Concerning the relapse cases by primary treatments: Although relapses were possible in any stages, they were more common with stage IIIB and IIIC cases. Those who achieved complete response after chemotherapy alone fewer relapse than those who had partial response after chemotherapy and there after had excision of residual tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

A phase II study of prophylactic intravesical chemotherapy with 4'-epirubicin in recurrent superficial bladder cancer: comparison of 4'-epirubicin and adriamycin.

Since intravesical recurrence of superficial bladder cancer (Ta, T1) after transurethral resection (TUR) is frequent, adjuvant therapy to reduce the recurrence rate has been extensively investigated. Although intravesical chemotherapy has been employed for 30 years or more, neither the exact effect on the bladder epithelium nor the optimal dose and administration schedule has yet been clarified. In recent years, several derivatives of Adriamycin (ADR) have been developed, and 4'-epirubicin (FARM) is one of them. This drug has been shown to have antitumor effects almost equal to those of ADR and to produce less toxicity when given systemically as chemotherapy. In an attempt to clarify the effect of intravesical FARM in the prevention of recurrence of superficial bladder cancer, we conducted a prospective randomized trial to compare the effects of equal doses of FARM and ADR given by intravesical instillation after TUR in cases of highly recurrent superficial bladder cancer. A total of 73 patients with recurrent superficial bladder cancer were randomized to receive TUR and either 30 mg FARM or 30 mg ADR by intravesical instillation every 2-4 weeks for 1 year. The prophylactic effect on recurrence and the toxic effects of these drugs were investigated. The current results show that FARM provides efficacy almost equal to that of ADR in the prevention of recurrence in these patients. However, FARM also caused almost the same local toxic effects (bladder irritation, among others) as ADR. On the basis of these preliminary results, FARM is surmised to be one of the agents as beneficial as ADR in the prevention of recurrence of superficial bladder cancer.

Computer imaging analysis of the correlation between intensities of glomerular immune-deposits and histopathology in patients with IgA nephropathy.

The relationship between the intensities of IgA, C3c, and C9 deposition in renal glomeruli and the severity of histopathologic injuries in patients with IgA nephropathy was examined using Microscope-Photometer 01K and a computer. Percentages of glomerular adhesion to Bowman's capsules, crescent formation, and glomerular sclerosis were calculated in the renal specimens. There was a significant correlation between the intensity of each C3c and C9 deposition in glomeruli and the degree of glomerular adhesion to Bowman's capsules and crescent formation in patients with IgA nephropathy. There was no significant correlation between the intensity of C3c or C9 deposition in glomeruli and the degree of glomerular sclerosis. No relationship was found between the intensity of IgA deposition in glomeruli and the degree of histopathologic injuries. The patients with negative or trace amounts of glomerular C3c deposits showed less severe glomerular injuries. Thus, the intensity of C3c and C9 deposition in glomeruli appears to be one of the critical factors responsible for the active progression of glomerular inflammatory process in patients with IgA nephropathy.

[A randomized trial of PVB, VAB-6, BVP regimen versus PEB chemotherapy in patients with disseminated testicular tumors].

During 2 years and 7 months from June, 1985 to December, 1987, a randomized multi-center trial of PVB, VAB-6, BVP regimen (group A) without etoposide versus PEB chemotherapy (bleomycin, etoposide and cisplatinum) (group B) was given to patients with disseminated testicular tumors. Of 34 patients registered, 10 patients were with minimal disease in stages IIA, IIIO and IIIA and 24 with extensive disease in IIB, IIIB2 and IIIC. Seminomas were found in 10 patients, while non-seminomatous tumors in 24. Among groups A and B, there was no statistical difference in clinicopathological profiles. A group patients were given either PVB, VAB-6 or BVP according to the physician's discretion. In groups A and B, 35% and 43% of the patients achieved complete response, and 45% and 50% achieved partial response, respectively. The difference in CR rates among both groups was not statistically significant even when calculated according to the stage or histologic grouping. Salvage treatments mainly with surgical resection of residual tumors after the chemotherapy, however, were more successful in group B (88%) than group A (61%). It appears likely that the higher response of induction chemotherapy in patients with extensive disease made the salvage surgery more successful in group B than in group A. The 3 year survival rate was 100% in group B, whereas it was 76% in group A. Although the incidence of myelosuppression and alopecia was significantly higher in group B, neuropathy was significantly more frequent in group A. From the above results, PEB seems to be a better induction chemotherapy than the conventional one for advanced testicular tumors.

Immunohistochemical studies on the localization of non-serum proteins detectable in human vaginal secretions.

Non-serum proteins in human vaginal secretions (HVS) were localized by an immunohistochemical method. A non-serum protein localized at the internal margin of the vaginal epithelium was considered to be an antigen-b, a main non-serum protein in HVS. As vaginal epithelium has no gland tissues, the production of this antigen is discussed.

[Phase II study of 5-FU tablet in bladder tumor].

A cooperative-phase II study of 5-FU tablet at a dose of 200 to 300 mg/day (b.i.d. or t.i.d) on 27 patients with bladder cancer was performed at Yokohama City University and other affiliated hospitals. The therapeutic responses were evaluated by Koyama-Saito's criteria in 24 out of 27 patients, and 3 PR, 2 MR, 16 NC and 3 PD were obtained. The overall response rate was 12.5% (3/24). All of the responders received 5-FU tablets at a daily dose of 300 mg. Side effects were found in 2 out of 26 patients (7.7%). These were slight gastrointestinal symptoms. These results suggest that 5-FU tablet is a useful drug for bladder cancer.

[A case of retroperitoneal fibrosarcoma].

A case of retroperitoneal fibrosarcoma is reported. An 81-year-old man was admitted to our hospital with gross hematuria. X-ray examination of intravenous pyelography, computed tomography and angiography revealed left hydronephrosis and left renal tumor. Excisional surgery was carried out. The tumor and left kidney were completely removed. Pathological diagnosis was retroperitoneal fibrosarcoma. Five months later, he died of lung and liver metastasis.

[Urachal transitional cell carcinoma invading the sigmoid colon].

We report a patient with urachal transitional cell carcinoma invading the sigmoid colon. The diagnostic and therapeutic aspects of this disease are discussed. The literature is also reviewed. This 42-year-old man was admitted with the chief complaint of micturition pain. Operation consisting of total cystourethrectomy, excision of the urachus together with the umbilicus, pelvic lymphadenectomy, ilial conduit plasty, sigmoidectomy, and colorectostomy was performed on June 3, 1986, after the diagnosis of urachal carcinoma invading the sigmoid colon. A tumor the size of a hen's egg was found at the dome of the bladder extending to the sigmoid colon. The urachus 8 cm in length and 2 cm in diameter extended from the site of the tumor to the umbilicus. Histologically the tumor was a transitional cell carcinoma Grade III. The urachus was composed of epithelioid and muscle layers, and its lower end was filled with necrotic inflammatory debris so that the canal was obliterated; tumor cells infiltrated the muscle layer. No metastasis was found in lymphnodes. Combination therapy of 5 fluorouracil, adriamycin and cis-platinum was given after operation. He has returned to work and is doing well without evidence of metastasis or recurrence now at 1 year after the operation.