A novel nonreceptor tyrosine kinase, Srm: cloning and targeted disruption.

We have isolated a novel nonreceptor tyrosine kinase, Srm, that maps to the distal end of chromosome 2. It has SH2, SH2', and SH3 domains and a tyrosine residue for autophosphorylation in the kinase domain but lacks an N-terminal glycine for myristylation and a C-terminal tyrosine which, when phosphorylated, suppresses kinase activity. These are structural features of the recently identified Tec family of nonreceptor tyrosine kinases. The Srm N-terminal unique domain, however, lacks the structural characteristics of the Tec family kinases, and the sequence similarity is highest to Src in the SH region. The expression of two transcripts is rather ubiquitous and changes according to tissue and developmental stage. Mutant mice were generated by gene targeting in embryonic stem cells but displayed no apparent phenotype as in mutant mice expressing Src family kinases. These results suggest that Srm constitutes a new family of nonreceptor tyrosine kinases that may be redundant in function.

Growth of mouse mammary glands after neonatal sex hormone treatment.

Mammary gland growth was examined in female mice immediately and during 1 month after neonatal treatment with sex hormones. BALB/c mice were given daily injections of hormones for the first 5 days after birth. As a criterion of growth, the number of ductal branchings was counted in stained wholemounts of the fourth (inguinal) pair of glands. At day 6, the growth of the glands was significantly inhibited in animals treated with 17 beta-estradiol (E) or diethylstilbestrol (DES); after treatment with progesterone (P) or androgens, no immediate effect was evident. At day 33, neonatal treatment with E stimulated growth, as did neonatal treatment with testosterone (T) or 5 alpha-dihydrotestosterone (5 alpha-DHT); treatment with a high dose of DES inhibited growth. Treatment with P or 5 beta-dihydrotestosterone had no effect. In conclusion, neonatal treatment with sex hormones has two distinct effects on the growth of mouse mammary gland: 1) inhibition by estrogens, observed immediately after treatment, and 2) stimulation by E, T, and 5 alpha-DHT, observed 4 weeks after treatment.

The adenovirus E1A domains required for induction of DNA rereplication in G2/M arrested cells coincide with those required for apoptosis.

Induction of apoptosis by adenovirus E1A in rodent cells is stimulated by wild type (wt) p53 but completely suppressed by mutated p53. The suppression is overcome by coexpression with Id proteins (Ids). The cells expressing E1A and Ids undergo apoptosis after accumulation in S phase, suggesting that S phase events are perturbed by E1A and Ids. The E1A domains required for induction of apoptosis, analysed by transfection with expression vectors for E1A, Ids and their mutants, followed by flow cytometry, reside in N-terminal (positions 17 - 38), CR1 and CR2 regions. Interaction of E1A with Ids requires the N-terminal and CR1 regions. The cyclin D1 promoter activity in S phase was reduced severely by E1A and this reduction is caused through CR1 and CR2 regions required for interaction with pRB. Analysis of DNA synthesis in G2/M arrested cells indicated that E1A is capable of inducing >4 N cells and this E1A-mediated DNA rereplication is enhanced by coexpression with Id-1H. The E1A domains required for induction of DNA rereplication coincide with those required for apoptosis.

Enhanced proliferative potential in culture of cells from p53-deficient mice.

Normal somatic cells are endowed with limited doubling potential in culture, and the process of immortalization is an inevitable step in neoplastic transformation of the cells. To examine the roles of p53 in this process, the cells of p53-deficient mice were examined for doubling potential. Fibroblast-like cells from a variety of tissues of these mice proliferated continuously without showing aging or crisis. The aneuploid cells overcome the population with passage, but cloning experiment indicated that chromosomal changes were not essential to this process. The enhanced proliferative potential in culture of cells from the p53-deficient mice was also observed in epithelial cells of lens, mammary glands and seminal vesicles and in neural precursor cells. Proliferation of bone marrow cells in response to stem cell factor was enhanced in long term culture, but not in in vitro colony assay; no permanent cell lines could be obtained. No effects of p53-deficiency were found in proliferation of cardiac muscle cells or hepatocytes.

Identification of a developmentally regulated gene in the mouse central nervous system which encodes a novel proline rich protein.

A full length cDNA whose corresponding mRNA is down-regulated during the mouse embryonic brain development was isolated. The cDNA contains a single long open reading frame which could encode a protein with relative molecular mass of 41 kDa. The predicted gene product contains long stretches of prolines towards the NH2-terminus, followed by a leucine/proline rich region. The cDNA probe detected a number of mRNA species in Northern blot analysis. The reverse transcriptase-polymerase chain reaction analysis of mRNA from adult mouse tissues indicated that heart and testis expressed this gene (named NDPP-1) at relatively high levels, while lower levels of mRNA were detected in a number of other tissues. Expression of NDPP-1 was also detected in embryonic carcinoma and pheochromocytoma cell lines, but not in fibroblasts. The cDNA hybridized to genomic DNA from several vertebrates species in Southern blot analysis indicating interspecies conservation of this gene. The interesting pattern of expression of the NDPP-1 gene during mouse brain development and the structure of its putative protein product indicate that this gene may play an important biological role in the development of mouse central nervous system.

Mouse nestin cDNA cloning and protein expression in the cytoskeleton of transfected cells.

Complementary DNA (cDNA) corresponding to mouse nestin intermediate filament protein, a specific marker for neural stem cells, was isolated and characterized. The complete sequence comprised 5983 base pairs encoding 1821 amino acids, and the deduced polypeptide was similar to rat (84%), hamster (73%), and human (62%) nestin. Southern blots showed that mouse nestin was a single-copy gene, and Northern blots detected a 6.0 kilobase mRNA transcript. When the cDNA was overexpressed as an enhanced green fluorescent fusion protein in COS7 cells, nestin immunoreactivity appeared in the filamentous cytoskeletal network. Accordingly, biologically active mouse nestin cDNA may offer an important new tool for stem cell research.

Stimulation of mammary epithelial cell growth in vitro: interaction of epidermal growth factor and mammogenic hormones.

A serum-free primary cell culture system was used to examine the direct effects and interactions of mammogenic hormones and epidermal growth factor (EGF) on the growth of mouse mammary epithelial cells. Epithelial cells were isolated by collagenase dissociation followed by Percoll gradient centrifugation and cultured within collagen gels in a mixture of Ham's F-12-Dulbecco's Minimum Essential Medium (1:1) containing insulin (10 micrograms/ml), crude soybean lecithin, trace elements, trypsin inhibitor, and antioxidants. Progesterone (P; 10(-6) - 10(-8) M) or ovine PRL (1 microgram/ml), in the absence of EGF, stimulated the growth of cells from mature virgin mice 2- to 4-fold over that of controls cultured in basal medium only. P and PRL synergized in stimulating growth 3- to 17-fold. 17 beta-Estradiol (10(-7) - 10(-10) M) alone did not stimulate growth or synergize with P and/or PRL. This lack of growth stimulation by 17 beta-estradiol was also observed in medium containing a low concentration of insulin (0.1 microgram/ml). EGF (10 ng/ml) alone stimulated growth to the same extent as the combination of P and PRL. EGF at 1, but not 10, ng/ml when combined with P and PRL could additively stimulate growth. Cells from midpregnant mice were less responsive than cells from virgin mice to the growth-stimulating effects of the combination of P and PRL (2-fold stimulation at most), but not to EGF (3- to 6-fold stimulation). Corticosterone, deoxycorticosterone, and aldosterone, but not cortisol, could synergize with PRL in stimulating the growth of cells from mature virgin mice. However, only deoxycorticosterone could stimulate growth in the absence of PRL. These results suggest that PRL, P, and adrenal corticoids may directly stimulate the growth of mouse mammary epithelial cells. The physiologically relevent adrenal corticoids, corticosterone and aldosterone, only potentiate the stimulatory effect of PRL. The hormonal stimulation of growth in vitro can be obscured by an optimum concentration (10 ng/ml) of EGF. The relative growth responses to mammogenic hormones and EGF may depend on the degree of differentiation of the cells.

Proliferation of mouse uterine epithelial cells in vitro.

Although estrogens can stimulate the growth of uterine epithelial cells in vivo, there is no clear effect of estrogens on the in vitro growth of epithelial cells from reproductive tract tissues; thus, we have established a defined culture system for mouse uterine epithelial cells. Pieces of uteri from immature CD-1 mice (21-23 days of age) were treated with trypsin, and the epithelial fragments were separated, enriched by Percoll gradient centrifugation, and seeded on collagen gels prepared from rat tail tendon. Initially, the cells were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's Medium supplemented with epidermal growth factor (EGF; 10 ng/ml), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), hydrocortisone (0.1 micrograms/ml), and vitamin A (10 ng/ml). The cells formed a monolayer on the collagen gel within 1-2 days, but with time, cells began to detach from the gel. Further studies revealed that the attachment and growth of these cells on collagen were markedly influenced by the calcium concentration. It was found that lowering the calcium concentration from 1.05 to 0.05-0.1 mM dramatically suppressed cell detachment; the number of cells doubled after 7 days of culture. Proliferation of uterine epithelial cells was enhanced by EGF, but not by fibroblast growth factor, platelet-derived growth factor, nerve growth factor, multiplication-stimulating activity, or somatomedin-C. The uterine epithelial cells exhibited a single class of high affinity binding sites for [125I]iodo-EGF (Kd, approximately 1.8 nM), with approximately 5 X 10(4) receptors/cell; binding was inhibited by EGF but not by the other polypeptides. This cell culture system will aid in our investigations on hormonal effects on the growth and differentiation of estrogen target cells.

Growth of mammary epithelial cells from neonatally sex hormone-exposed mice in serum-free collagen gel culture.

Neonatal sex hormone treatments are known to cause an increase in mammary tumors in female mice with expressed mammary tumor virus (MTV). The growth of mammary epithelial cells from mice treated neonatally with sex hormones was studied in response to growth-stimulatory factors in a serum-free collagen gel culture system which sustains the growth of normal mammary epithelial cells. Animals were treated with hormones or oil-vehicle for the first 5 days after birth. Cells from control mice at 2 and 3 months of age showed a maximal growth response to insulin at 5-10 micrograms/ml and LiCl at 5-20 mM. Cells responded to epidermal growth factor at all concentrations used (1, 10 and 50 ng/ml). In contrast, mammary epithelial cells from mice treated neonatally with estrogen (estradiol and diethylstilbestrol (DES] showed a reduced growth response to the growth factors tested.

Extracorporeal urinary bypass for malignant ureteral obstruction.

Extracorporeal urinary bypass was attempted in a patient with malignant ureteral obstruction. A nephrostomy was drained into the bladder by connecting the tube to a cystostomy catheter. This method made the patient free from a collecting bag without any significant complications. This method may improve the quality of life of patients with malignant ureteral obstruction, when their bladder is intact.

Cerebellar cell lines established from a p53-deficient adult mouse.

p53-Deficient mice are prone to develop spontaneous tumors [L.A. Donehower, M. Harvey, B.L. Slagle, M.J. McArthur, C.A. Montgomery Jr. , J.S. Butel, A. Bradley, Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors, Nature 356 (1992) 215-221], but brain neoplasms are rare and difficult to culture in vitro. Here we describe cloning and long-term culture of heterogeneous neural cell lines from a multifocal cerebellar neoplasm that arose in an adult p53-/- mutant mouse. These lines may be useful for studies of neoplastic transformation and cell lineage in cerebellar development.

A possible role of the nestin protein in the developing central nervous system in rat embryos.

Rat embryos at the head-hold stage (Slc:SD strain; 9.5 days of gestation) were cultured for 48 h in rat serum with the anti-nestin peptide antiserum. The antiserum identified a single band in Western blots of the tissue extracts from rat embryos and stained the cells from the neural tube, migrating neural crest, and somites immunohistochemically. The antiserum-treated embryos appeared to develop normally for the most part. However, histological observation disclosed that the ventral portion of the neural tube was deformed. The cells in the deformed portion did not show the elongated shape but were round. These round cells tended to crowd near the ventricular surface, and a gap was observed between the original pial surface and cells arranged in the most pialward region. The penetration of the anti-nestin peptide antibody into the embryos from the culture medium was confirmed by visualization of the penetrated antibody using biotinylated anti-rabbit IgG antibody raised in goats and Texas red-conjugated streptavidin. These results indicate that the nestin protein plays an important role in the organization or the maintenance of neuroepithelial cells of the elongated shape spanning the neural tube from the luminar to the pial side.

Symptomatic accessory lobe of the liver associated with hyperthyroidism.

A case of symptomatic accessory lobe of the liver occurred in a 15-year-old Japanese girl with hyperthyroidism. The patient presented with acute abdominal distress; at operation, a twisted necrotic mass of the accessory lobe of the liver was found. A review of the literature failed to show a previously reported instance in a child.

[A case of exohepatic pedunculated hepatocellular carcinoma suspected of adrenal tumor].

A case of exohepatic pedunculated hepatocellular carcinoma that was clinically diagnosed as nonfunctioning adrenal tumor is reported. A 66-year-old man was admitted to our hospital for further examination of unstable angina pectoris. Abdominal echogram and CT scan revealed a large tumor in the right retroperitoneal space. Selective right renal arteriography demonstrated that the tumor was fed by the capsular branch of right renal artery and the right adrenal artery. Adrenal function was normal. Preoperative diagnosis of right nonfunctioning adrenal tumor was made. On operation we found that the tumor was pedunculated from the liver and adhered massively to both right kidney and vena cava. The tumor and right kidney were removed. A histopathological examination demonstrated well differentiated hepatocellular carcinoma (Edmondson's grade II type).

[Rectal metastasis of prostatic cancer causing annular stricture: a case report].

A 61-year-old man diagnosed with poorly differentiated adenocarcinoma of the prostate (T4 NxM0, stage C) underwent endocrine therapy. The reduction of the tumor was recognized but soon annular rectal stricture appeared. In spite of the subsequent chemotherapy, symptoms aggravated. Then total pelvic exenteration and colostomy were performed. Prostate was easily separated from the rectal wall and the tumor continuity was not proved. Immunohistochemical inspection indicated that the origin of the tumor cells of the rectum was the prostate. Histopathological examination of the rectum using the step section method showed no trace of cancer invasion but many cancer cells in the intramural lymphatic duct. We concluded that adenocarcinoma of the prostate metastasized to the rectum by way of lymphatic flow and caused the annular stricture of the rectum.

[Spontaneous peripelvic extravasation associated with primary ureteral tumor: a case report].

A case of spontaneous peripelvic extravasation associated with primary ureteral tumor is reported. A 56-year-old woman presented with left flank pain. Excretory urogram and abdominal computed tomographic (CT) scan demonstrated left hydronephrosis with extravasation of contrast materials around the renal pelvis. Retrograde pyelogram showed the filling defect in the left upper ureter. Under diagnosis of ureteral tumor, total nephroureterectomy was performed. Histological findings revealed transitional cell carcinoma. 80 cases of spontaneous peripelvic extravasation in Japan were reviewed and discussed briefly.

[Clinical statistics on inpatients and operations at the Department of Urology, the Center for Adult Diseases, Osaka, 1984-1988].

Statistical observation on inpatients and operations at our department between January 1984 and December 1988 revealed the following results: 1) The total number of inpatients was 1962 (male: 1658, female: 304). The most frequent diseases were bladder cancer (30.0%), benign prostatic hypertrophy (19.2%), prostatic cancer (10.6%) and renal cancer (6.7%). 2) The total number of operations was 1699. The most frequent operations were transurethral resection (TUR) of bladder tumor (22.8%), TUR prostate (20.7%), TUR biopsy (6.5%) and total cystectomy (5.4%).

[Production of monoclonal antibodies against human bladder cancer cells and application to immunohistochemical analysis of bladder cancer].

Two hybridomas secreting two monoclonal antibodies IgG1 B1.4 and IgG2a B1.6 were obtained by immunizing BALB/c mice with human bladder cancer cell line EJ-1. In immunohistochemical staining of cryopreserved tissues, B1.4 reacted with 0 of 9 grade 1 TCC, 6 of 11 grade 2, all of 6 grade 3 and five metastatic specimens. The antigen recognized by B1.4 was not expressed by normal urothelial cells but were expressed by vascular endothelial cells and muscle of tunica media. The target antigen of B1.6 was expressed by normal urothelial cells and all grade of TCC. In this study, it was demonstrated that poorly differentiated bladder cancer and metastatic specimens of bladder cancer express a vascular carbohydrate antigen. Taking the escape mechanism of immune surveillance, into consideration, it is possible that the antigen recognized by B1.4 is an indicator of metastatic potential of bladder cancer.

[Upper urinary tract tumors following bladder cancer].

We treated in total 795 patients with primary transitional cell carcinoma of the urinary bladder between April, 1964 and December, 1988. Eighteen patients of them had upper urothelial cancer during the follow-up period. Thirteen of the 18 patients had received transurethral resection for the initial bladder cancer, while 3 total cystectomy and 2 segmental resection. The over-all incidence of bladder cancer patients who subsequently developed upper urinary tract tumors was 2.3 per cent. The interval between initial treatment of the bladder cancer and treatment for the upper urinary tract tumor ranged from 2 to 74 months (median 20 months). The five-year survival rate after treatment for the upper urinary tract tumor was 31.7 per cent. We conclude that the following are high risk patients for development of upper urinary tract recurrences: 1) patients with bladder cancer near orifices, 2) patients with recurrent bladder cancer under bladder preserving treatment for a long time, 3) patients with G2 multifocal bladder cancer.

[VM-26 and VP-16 salvage therapy for refractory germinal testicular cancers].

Thirteen evaluable patients with germinal testicular cancers failing to be cured with first-line therapy (refractory) were treated by salvage chemotherapy. Ten patients received salvage chemotherapy with VM-26 (50 mg/m2, twice a week X 6 weeks) and cisplatin (CDDP, 20 mg/m2 for 5 consecutive days every 3 weeks for 3-4 times) (P-VM), 3 patients were also treated by radiation therapy, and 3 patients received VP-16 (100 mg/m2) and CDDP (20 mg/m2) (P-VP), all given daily for 5 consecutive days every 3-4 weeks for 4-5 courses. Of 13 evaluable patients, 6 (46%) had complete response (CR) (three cases were also treated with radiation therapy), 4 (31%) achieved partial response (PR), and 3 (23%) had no response. Limited to 7 patients treated with only P-VM therapy, there were 3 (43%) CR and 4 (57%) PR. Nine patients (69%) remained alive and were continuously disease free 18 to 84 months (median 48 months). Hematologic toxicity was severe, but with no death related to sepsis. Salvage chemotherapy with VM-26 or VP-16 and cisplatin offers potentially curative treatment to patients with refractory testicular cancer. The addition of radiation therapy to salvage chemotherapy was also effective.