Efficient and ligand-dependent regulated erythropoietin production by naked dna injection and in vivo electroporation.

The development of an in vivo gene transfer approach to deliver physiologic levels of recombinant proteins to the systemic circulation would represent a significant advance in the treatment of protein deficiencies-disorders. However, the ability to regulate transgene expression will become paramount for safety and efficacy in gene transfer therapy. We have described the construction of an efficient and ligand-dependently regulated erythropoietin (EPO) production system using naked plasmid and in vivo electroporation. Two plasmids, one encoding the chimeric GeneSwitch protein and the other encoding an inducible transgene for human EPO, were developed. Modulation of the level of secretion of EPO into the serum was achieved by intraperitoneal administration of mifepristone (MFP). Rats were divided into 4 groups: one group received EPO plasmid with MFP for 30 days, a second group received with EPO plasmid MFP for 9 days, a third group received EPO plasmid without MFP, and a fourth group received control plasmid. A pair of electrodes was inserted into the muscle of the right thigh and 100 micrograms of each plasmid was injected. In vivo electrporation (8 times at 100 V for 50 milliseconds) was performed. The presence of vector-derived EPO mRNA was detected by reverse transcriptase-polymerase chain reaction only in the EPO and MFP(+) group. The hematocrit levels increased continuously from the preinjection level of 42.7% to 53.8% on day 28 in the EPO and MFP(+) group. The serum EPO levels increased only in the EPO and MFP(+) group. There was no significant change in hematocrit levels and EPO levels in the EPO and MFP(-) group. These results demonstrate that EPO gene transfer with the GeneSwitch system by in vivo electroporation is a useful procedure for efficient and drug-dependent regulated delivery of EPO.

Hyperosmolality activates Akt and regulates apoptosis in renal tubular cells.

BACKGROUND: The novel serine-threonine kinase Akt is a critical enzyme in cell survival. We investigated the roles of the Akt pathway and apoptotic signals in (1) Madin-Darby canine kidney (MDCK) cells in a hyperosmotic condition in vitro and (2) in the inner medulla of dehydrated rat in vivo. METHODS: The in vivo experiments were performed in 24- and 48-hour water-restricted rats. Hyperosmolality-stimulated Akt phosphorylation was examined in MDCK cells. Phosphatidylinositol 3-kinase (PI3-K) inhibitors, the dominant-negative mutant of PI3-K, the dominant-negative mutant of Akt, and the dominant-active form of Akt were used to examine the roles of the PI3-K/Akt pathways in renal tubular cell apoptosis. RESULTS: The amount of phosphorylated Akt protein was increased in the inner medulla of dehydrated rats. Hyperosmolality induced by the addition of NaCl, urea, and raffinose phosphorylated Akt in MDCK cells in an osmolality-dependent manner. PI3-K inhibitors and the dominant-negative mutant of PI3-K inhibited the hyperosmolality-induced phosphorylation of Akt. Raising the media osmolality from a normal level to 500 or 600 mOsm/kg H2O final osmolality elicited apoptotic changes such as nucleosomal laddering of DNA and an increment of caspase-3 activity and increased activity in the cell death enzyme-linked immunosorbent assay. Dominant-active Akt prevented the mild hyperosmolality-induced apoptosis, while inhibition of the PI3-K/Akt pathways promoted apoptosis. CONCLUSION: The Akt pathway is activated by hyperosmolality in vitro and in vivo, and activation of Akt prevents the mild hyperosmolality-induced apoptotic changes in MDCK cells. PI3-K/Akt pathways are involved in a hypertonic condition that confers the balance between cell survival and apoptosis.

Overexpression of protein kinase G using adenovirus inhibits cyclin E transcription and mesangial cell cycle.

The cGMP-cGMP-dependent protein kinase (protein kinase G) system plays an important role in the pathogenesis of mesangial proliferative glomerulonephritis. However, the molecular mechanisms of the inhibitory effects of the cGMP-protein kinase G system in the cell cycle progression of mesangial cells are not well known. To determine the inhibitory pathway of cGMP-protein kinase G in cultured mesangial cells, we investigated the effects of cGMP- and adenovirus-mediated overexpression of protein kinase G on the promoter activities of cyclin E, cyclin D1, and cyclin A. 8-Bromo-cGMP (8-BrcGMP) and overexpression of protein kinase G reduced [(3)H]thymidine uptake, reduced the numbers of cells in S and G(2)/M phases, and decreased the phosphorylation of retinoblastoma (Rb) protein. 8-BrcGMP (10(-3) M), protein kinase G adenovirus (Ad-cGKIbeta; 10(10) plaque-forming units/ml), atrial natriuretic peptide (ANP), and C-type natriuretic peptide (CNP) inhibited the promoter activity of cyclin E to 49, 57, 77, and 78%, respectively. On the other hand, the promoter activities of cyclin D1 and cyclin A were not changed significantly. In Western blot analysis, 8-BrcGMP, Ad-cGKIbeta, ANP, and CNP also inhibited cyclin E protein expression dose and time dependently. The p44/p42 mitogen-activated protein kinase (MAPK) kinase 1-p44/p42 MAPK had no effect on cyclin E promoter activities, and the cGMP-protein kinase G pathway did not change MAPK activity. In conclusion, our findings suggest that the reduction of the cyclin E promoter activity that downregulates G(1)/S transition plays a dominant role in the cGMP- and protein kinase G-induced inhibition of mesangial cell proliferation.

Glucocorticoids stimulate p21(CIP1) in mesangial cells and in anti-GBM glomerulonephritis.

BACKGROUND: Glucocorticoids are widely used for the treatment of glomerulonephritis, but the mechanism of cell cycle inhibition by glucocorticoids is poorly understood at a molecular level. METHODS: The effects of dexamethasone on cell cycle progression were examined in rat mesangial cells. To investigate the mechanisms of cell cycle inhibition by dexamethasone, we transfected the -2.3 kb p21(CIP1) promoter-CAT construct to mesangial cells using an electroporation METHOD: We also examined whether glucocorticoids stimulate the expression of p21(CIP1) and inhibit cell proliferation in glomeruli of anti-glomerular basement membrane (GBM) glomerulonephritis in rats. RESULTS: Dexamethasone inhibited 3H-thymidine uptake and the percentages of S and G2/M phases in rat mesangial cells. Dexamethasone stimulated CAT activity of the p21(CIP1) promoter 4.5-fold. Deletion analysis of the p21(CIP1) promoter revealed that the glucocorticoid-responsive region (GRE) is present between -1.4 and -1.1 kb upstream of the transcription initiation site. Dexamethasone inducibility of p21(CIP1) promoter activity requires the presence of the C/EBP alpha DNA binding site in the GRE of the p21(CIP1) promoter and C/EBP alpha protein. Intravenous injection of anti-GBM antibody caused mesangial proliferation, crescent formation, and proteinuria in rats. Ten days of administration of prednisolone (1 mg/kg/day) reduced proteinuria and inhibited mesangial cell proliferation and crescent formation. The glomerular-sieving method revealed that prednisolone increased p21(CIP1) expression in glomeruli. CONCLUSION: These data suggest that the cell cycle arrest of mesangial cells is mediated by a functional link between the glucocorticoid receptor and the transcriptional control of p21(CIP1) not only in vitro but also in vivo. Our observations provide new insights into the molecular mechanisms of glucocorticoid action in glomerulonephritis.

Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation.

ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine- and stress-induced apoptosis. However, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced apoptosis. Overexpression of ASK1DeltaN, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 (-/-)) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1-induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9-dificient (Casp9 (-/-)) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute apoptosis mainly by the mitochondria-dependent caspase activation.

Roles of E2F1 in mesangial cell proliferation in vitro.

UNLABELLED: Roles of E2F1 in mesangial cell proliferation in vitro. BACKGROUND: The proliferation of mesangial cells is a common feature of many glomerular diseases. E2F transcription factors play an important role in the regulation of the cell cycle. However, the regulation of the mesangial cell cycle and the participation of the E2F family (E2F1 through E2F5) in mesangial cells have not been clarified. Therefore, we investigated the roles of the E2F family in the mesangial cell cycle. METHODS: To elucidate the importance of the E2F family, we investigated the mesangial cell cycle by examining the cell count and thymidine incorporation, and compared it with the protein expression of E2F. Using adenovirus-mediated gene transfer, the cell cycle and apoptosis were examined by measurement of thymidine incorporation, flow cytometry, and caspase 3 activity. We also studied the interaction between E2F1 and G1 cyclins by promoter assay, Western blotting, and CDK kinase assay. RESULTS: E2F1 increased 20-fold in G1/S phase transition. E2F1 overexpression facilitated the mesangial cell cycle and later induced apoptosis. Furthermore, E2F1 overexpression increased the promoter activities and protein expressions of G1 cyclins, cyclin D1, cyclin E, cyclin A. The up-regulation of G1 cyclins contributed to the activation of CDK4 and CDK2. CONCLUSIONS: In mesangial cells, we conclude that E2F1 plays an important role in G1/S phase transition and in apoptosis. E2F1 regulates the mesangial cell cycle through two distinct pathways. First, E2F1 directly transcribes genes that are necessary for DNA synthesis, and second, it promotes cell cycle progression via the induction of G1 cyclins.

Regulation of the G1/S transition phase in mesangial cells by E2F1.

It has been established that E2F transcription factors are essential for the regulation of the cell cycle. The E2Fs play an important role in G1/S transition phase, as they regulate the activation of several genes whose products are required for DNA synthesis. E2Fs bind to the retinoblastoma protein family and their transcriptional activities are suppressed in the G0 and early G1 phases. The E2F family consists of a group of five closely related proteins (E2F1 through E2F5). Proliferation of the mesangial cell is a common feature of many glomerular diseases, but the regulation of mesangial cell cycle has not been clarified, nor has the participation of the E2F family in mesangial cells. To elucidate the mechanisms of G1/S transition phase in mesangial cells, we investigated the roles of the E2F family in the mesangial cell cycle. In primary cultured mesangial cells, the protein expression of E2F1 through E2F3 was induced by fetal calf serum (FCS) stimulation. E2F1 especially was strongly induced by mitogenic stimulation. The E2F4 protein was abundantly expressed in the quiescent state and was slightly increased by FCS stimulation. We considered E2F1 to be representative of the E2F family, and used adenovirus-mediated gene transfer to investigate the function of E2F1 to show that overexpression of E2F1 promoted cell cycle progression as measured by a flow cytometer. Furthermore, we investigated the effect of E2F1 overexpression to cyclin D1 and cyclin E expression. Because we previously reported that the regulation of G1 cyclins is a key factor in the G1/S transition phase in mesangial cells, we showed that overexpression of E2F1 induced protein expression of cyclin D1 and cyclin E and increased promoter activity. Thus, we conclude that E2F1 plays an important role in the G1/S transition phase and acts on the mesangial cell cycle through two distinct pathways: (1) E2F1 directly transcribes an S-phase gene, and (2) E2F1 promotes cell cycle progression via the induction of cyclin D1 and cyclin E.

Regulation of cyclin D1 expression and cell cycle progression by mitogen-activated protein kinase cascade.

Mitogen-activated protein kinases (MAPKs) have been shown to play an important role in transducing extracellular signals into cellular responses. The classic MAPK pathway is commonly activated by growth factors and has been shown to play a crucial role in cell proliferation. Transforming growth factor-beta (TGF-beta)-activating kinase-1 (TAK1) is a novel MAPK kinase kinase that is reported to stimulate the MKK6-p38K pathway. To elucidate the functional roles of the TAK1 pathway, we transfected its constitutive active form (TAKdN) and negative form (TAKK63W) to LLC-PK1 cells. TAKdN stimulated MKK6 phosphorylation and p38K activity and inhibited the percentages of the S and G2/M phases. TAKK63W, the constitutive negative form, reduced TGF-beta-stimulated MKK6 phosphorylation and p38K activity and increased the percentages of the S and G2/M phases. The cyclin D1 protein level is reduced by the TAK1 pathway. We also examined the effects of the TAK1 pathway on cyclin D1 promoter-luciferase assay. The overexpression of TAKdN or p38K inhibited cyclin D1 promoter activity. In contrast, overexpression of the active form of MKK1, the classic MAPK-activator, MKK1 increased cyclin D1 promoter activity and protein level, as well as the percentages of S and G2/M phases.

TGF-beta-activating kinase-1 inhibits cell cycle and expression of cyclin D1 and A in LLC-PK1 cells.

BACKGROUND: Transforming growth factor-beta (TGF-beta) is known to play an important role in the pathophysiology of renal tubular disease. Researchers have recently identified a novel mitogen-activated protein kinase kinase kinase (MAPKKK), TAK (TGF-beta activated kinase)1, which stimulates the MKK3/6-p38K pathway. The purpose of our study was to investigate the functional role of the TAK1-MKK3/6-p38K pathway and classical MAPK cascades in the progression of the cell cycle in renal tubular cells. METHODS: The constitutive active form and negative form of TAK1 (TAK1dN and TAK1K63W, respectively), and active and negative forms of the p42/44 MAPK-activator, MKK1 (S222E and S222A, respectively) were transfected to LLC-PK1 cells. Western blot analyses and promoter-luciferase assay of cyclins D1, D2, D3, E, and A were performed, and cell cycle progression was analyzed by FACS scan. RESULTS: TAK1dN stimulated MKK6 and p38K activity and inhibited the percentage of the S and G2/M phases. TAK1K63 W inhibited TGF-beta-stimulated MKK6 and p38K activity. Cyclin D1 and cyclin A protein levels and promoter activities were negatively regulated by TAK1dN. In contrast, overexpression of the active form of p42/44 MAPK-activator, MKK1, increased cyclin D1 and A promoter activity and protein levels. CONCLUSION: The growth-inhibitory effects of TGF-beta are at least partially mediated by the TAK1-MKK6-p38K pathway. Cyclin D1 and A promoter activity and cell cycle progression in renal tubular cells are negatively regulated by the TAK1-MKK6-p38K pathway and positively regulated by the MKK1-p42/44MAPK pathway.

Cell cycle inhibitors (p27Kip1 and p21CIP1) cause hypertrophy in LLC-PK1 cells.

BACKGROUND: Angiotensin II has been reported to induce renal tubular hypertrophy, but the mechanisms of this hypertrophy are not well known. We evaluated the roles of cyclin-dependent kinase (CDK) inhibitors in renal tubular hypertrophy. METHODS: To elucidate whether CDK inhibitors cause renal tubular hypertrophy, we produced adenovirus vectors containing coding sequences of the CDK inhibitors p27Kip1 (AxCAp27), p21CIP1 (AxCAp21), and p16INK4 (AxCAp16), and we investigated the effect of these gene transfers on epidermal growth factor (EGF)-induced proliferation in LLC-PK1 cells. We evaluated the cell cycle and hypertrophy by measurements of the [3H]-leucine and [3H]-thymidine incorporation, the protein:DNA ratio, flow cytometry, and CDK4 and CDK2 kinase assays. RESULTS: AxCAp27 and AxCAp21 caused significant increases in [3H]-leucine incorporation and the protein:DNA ratio but did not change the [3H]-thymidine incorporation. Conversely, AxCAp16 inhibited EGF-stimulated [3H]-thymidine incorporation but did not change the [3H]-leucine incorporation. AxCAp27, AxCAp21, and AxCAp16 all inhibited EGF-stimulated CDK4 kinase activity (to 15.6, 14.1, and 21.9% of control, respectively). Forward light-scatter analysis demonstrated that AxCAp27 and AxCAp21 increased the cell size but that AxCAp16 effected no change in cell size. CONCLUSION: These findings suggest that p27Kip1 and p21CIP1 may play an important role in hypertrophy of renal tubule cells by reducing pRb phosphorylation. On the other hand, p16INK4 was not found to cause hypertrophic changes in EGF-treated LLC-PK1 cells.

Inducible nitric oxide synthase can be induced in the absence of active nuclear factor-kappaB in rat mesangial cells: involvement of the Janus kinase 2 signaling pathway.

The contribution of nuclear factor-kappaB (NF-kappaB) and interferon-gamma (IFN-gamma) signaling to nitric oxide generation is not completely understood. The effect of NF-kappaB release and its inhibition on nitrite production and the involvement of Janus kinase 2 (JAK2) in inducible nitric oxide synthase (iNOS) induction were investigated. The following assays were performed. (1) Nitrite produced by rat mesangial cells in primary culture was measured in incubations with tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS), with or without IFN-gamma. Cells were stimulated with TNF-alpha or LPS plus IFN-gamma in the presence of NF-kappaB inhibitors, herbimycin A (HerA), or the more specific JAK2 inhibitor AG490. (2) Immunoblotting was performed against the p65 and p50 subunits of NF-kappaB and iNOS. (3) Electrophoretic mobility shift assays were performed against NF-kappaB in the presence of NF-kappaB inhibitors or AG490. (4) iNOS promoter activity was measured in the presence of AG490 or JAK2 antisense oligonucleotides. TNF-alpha or LPS alone did not induce nitrite production, but with IFN-gamma these compounds did induce nitrite production. Pyrrolidine dithiocarbamate (PDTC), N-acetyl-L-cysteine, dexamethasone (Dex), HerA, and AG490 partially inhibited LPS/ IFN-gamma- or TNF-alpha/IFN-gamma-induced nitrite production. p65 was inhibited by the three NF-kappaB inhibitors described above, whereas p50 was not. PDTC and Dex completely inhibited the p65/p50 heterodimer, but HerA and AG490 had little effect on p65/p50. AG490 and JAK2 antisense oligonucleotides suppressed iNOS promoter activity. It can be concluded that (1) iNOS can be induced without active NF-kappaB; (2) Dex, acetylsalicylic acid, and PDTC inhibit only p65; and (3) JAK2 is involved in iNOS induction, and the contribution of JAK2 to nitrite production is greater than that of NF-kappaB.

Mitogen-activated protein kinase cascade and transcription factors: the opposite role of MKK3/6-p38K and MKK1-MAPK.

Recent studies have revealed that mitogen-activated protein kinase (MAPK) consists of at least three subfamilies, namely classical MAPK (also known as ERK), stress-activated protein kinase/c-Jun N-terminal kinase (JNK) and p38 kinase. TGF-beta-activating kinase (TAK)-1 is a novel MAPKKK which is reported to stimulate p38K and/or the JNK pathway. To elucidate the functional roles of the TAK1 pathway, we transfected its constitutive active form (TAK1dN) and negative form (TAK1K63W) into LLC-PK1 cells. TAKdN inhibited [3H]thymidine uptake and reduced the percentages of S and G2/M phases. TAK1K63W ameliorated the inhibitory effects of TGF-beta on [3H]thymidine uptake and increased the percentages of S and G2/M phases. Western blot analysis demonstrates that the level of cyclin D1 protein was regulated negatively by overexpression of TAK1dN. Moreover, overexpression of TAK1dN inhibited cyclin D1 promoter activity. In contrast, constitutive active MKK1, the classical p42/44 MAPK activator, increased cyclin D1 promoter activity and level of protein. Overexpression of the active form of MKK1 increased [3H]thymidine uptake, while the inactive form decreased the uptake. In conclusion, cyclin D1 promoter activity and cell cycle progression are regulated negatively by the TAK1 pathway and positively by the classical MAPK pathway.

Lovastatin inhibits mesangial cell proliferation via p27Kip1.

Mesangial cell proliferation is a key feature of glomerulonephritis. The hydroxymethylglutaryl-coenzyme A reductase inhibitor lovastatin is known to inhibit cell cycle progression. To determine the inhibitory mechanisms of mesangial cell proliferation by lovastatin, the cyclin-dependent kinase (CDK) activity, and expression of CDK inhibitor (p27Kip1, p21Cip1, and p16INK4) mRNA and protein were measured. Lovastatin inhibited phosphorylation of retinoblastoma protein and mesangial cell proliferation dose dependently. Lovastatin increased the p27Kip1 protein level but produced no changes in the abundance of the p27Kip1 mRNA level both in the presence and absence of mitogens. Treatment with lovastatin revealed the increment of both CDK2- and CDK4-bound-p27Kip1. The experiment using antisense oligonucleotide against p27Kip1 showed significant amelioration of lovastatin-induced cell cycle arrest. Lovastatin reduced both platelet-derived growth factor-stimulated CDK2 and CDK4 kinase activities. In conclusion, lovastatin inhibited mesangial proliferation via translational upregulation or impairment of p27Kip1 protein degradation. Lovastatin serves as a potential therapeutic approach to mesangial proliferative disease.

Cyclins and the cyclin-kinase system--their potential roles in nephrology.

Our understanding of the cell-cycle mechanisms has progressively advanced in the past few years. Cyclins and cyclin-dependent kinases play major roles as positive cell-cycle regulatory proteins and CDK inhibitors; while the Kip family and INK4 family are negative regulatory proteins in mesangial cells and renal tubular cells. An understanding of the cell cycle is essential for the rational design of novel pharmacotherapeutic approaches to suppress the excessive proliferation of mesangial cells in glomerular disease and hypertrophic tubular disease.

Cyclin D1, p16, and retinoblastoma gene regulate mitogenic signaling of endothelin in rat mesangial cells.

To elucidate the mechanisms by which endothelin (ET)-1 induces proliferation of mesangial cells, we investigated the involvement of the first gap phase of the cell cycle (G1) cyclin, cyclin-dependent kinase 4 (CDK4) activity, and the retinoblastoma gene product (pRb) in ET-1-stimulated cell cycle progression. In the present study, ET-1 stimulated CDK4 activity and cell cycle progression via ET A-type receptors and induced cyclin D1 mRNA and protein expression in rat mesangial cells. We also found that ET-1 stimulation of mesangial cell proliferation was inhibited by antisense oligonucleotides directed against cyclin D1 and by overexpression of a nonphosphorylatable form of pRb. To investigate the functional roles of p16INK4 and p21cip1 in ET-1-stimulated mesangial cell proliferation, we used adenovirus-mediated gene transfer. Endothelin-1-stimulated [3H]-thymidine incorporation, CDK4 kinase activity, and the percent of cells in S phase were found to be significantly inhibited by overexpression of p16INK4 and slightly inhibited by overexpression of p21cip1. Thus, ET-1 induced cyclin D1 expression and stimulated CDK4 activity and cell cycle progression via the A-type receptor in rat mesangial cells. These effects were regulated by the expression of cyclin D1, p16INK4, p21cip1, and phosphorylatable form of pRb. The results of the present study provide the basis for further investigation of basic and therapeutic approaches towards mesangial proliferative diseases.

Gastric small cell carcinoma with squamous and neuroendocrine differentiation.

A rare gastric carcinoma containing diverse components, that is, neuroendocrine (small cell carcinoma), squamous and gland-like elements in an 82 yr old woman is described. Radiologic examination revealed a large ulcerated tumor, and a Borrmann type II tumor, 6.5 x 5 cm, was found in the resected stomach. Histologically, the tumor was mainly composed of small cells with hyperchromatic nuclei and scant cytoplasm. Argyrophilic granules were seen in these cells. There were also scattered foci of large cells with features of squamous cells, and many intermediate cells with oncocytic cytoplasm. The small cancer cells were positive for chromogranin A and neuron specific enolase. Squamous cell nests were positive for high molecular cytokeratin (CK), and intermediate cells were positive for low molecular CK. Electron microscopic examination revealed secretory granules in the small cells and tonofilaments in the squamous cells. This tumor might have originated from the pluripotential stem cell in the gastric epithelium.

Dieulafoy's ulcer associated with the tortuous caliber persistent arteries: report of three cases.

Many papers have reported that Dieulafoy's ulcer is one of the notorious causes of gastric hemorrhage. Three cases of shallow subfundic ulcers with massive bleeding are reported. The resected specimens have demonstrated that elevated caliber-persistent artery (CPA), a branch of the left gastric artery with few anastomoses, in the base of the ulcer has tortuous penetration from the serosa to submucosa, showing patchy, eccentric intimal fibroelastosis. These findings of CPA are almost the same in both anterior and posterior walls, namely both the ruptured and contralateral sides. Thus, morphogenesis of the ulcer may have originated from anatomical deviation, which is related to regional hypertension aggravated by longterm peristalsis, as well as aging.

Phyllodes tumor (cystosarcoma phyllodes) of the breast. A clinicopathologic study of 45 cases.

The correlation between the histology and biologic behavior of phyllodes tumor was studied in order to establish the histologic criteria of this tumor, particularly its malignant counterpart. A preliminary analysis of five tumors with metastases revealed that a single essential indicator of malignant potential was the presence of a disproportionate overgrowth of the stromal elements at the expense of the ductal element. The presence or absence of such stromal overgrowth in the tumor showed a positive correlation with the degree of stromal atypism and the rate of mitoses. Seventeen out of 45 tumors were determined to be malignant, on the basis of our criteria. The arrangement of the stromal component in malignant phyllodes tumors was usually one of the particular sarcomatous types, with or without the focal areas of one of the specified types of sarcoma. On the other hand, 24 tumors recurred locally but never metastasized, and were preferentially interpreted to be benign phyllodes tumor. Histologically, these tumors had a general architecture resembling that of fibroadenoma but were characterized by high cellularity expressed by the distinct formation of bundles or fascicles of stromal tumor cells. The remaining four tumors seemed to form a borderline phyllodes tumor subset. A follow-up study revealed that, irrespective of the mode of therapy, the outcome of patients with phyllodes tumor was well correlated with the histologic criteria we proposed.

Carcinosarcoma of the gallbladder. Report of a case and review of the literature.

An extremely rare case of carcinosarcoma of the gallbladder is reported. The tumor presenting as a large, soft polypoid growth was microscopically characterized by an admixture of adenocarcinomatous and sarcomatous elements, the latter containing specific heterologous mesenchymal elements such as osteoid, cartilage, and rhabdomyoblasts. The cytoplasm of neoplastic rhabdomyoblasts offered unequivocal cross striations and was strongly positive for myoglobin, determined by the immunoperoxidase procedure. Metastatic tumors in the liver also exhibited a dual composition of adenocarcinoma and rhabdomyosarcoma. This appears to be the second documented case of this type of neoplasm with the rhabdomyosarcomatous component. The clinical settings and morbid prognosis were similar to events in cases of carcinoma of the gallbladder.

Invasive lobular carcinoma of the breast. An analysis of 29 cases.

Twenty-nine cases of invasive lobular carcinoma were analyzed, based on three aspects of the histology: 1) cellular features such as a monotonous proliferation of uniform small cells, 2) a single file or targetoid arrangement, and 3) loss of cell cohesion or dissociation of tumor cells. Twenty-four tumors which fulfilled these three criteria were appraised as cases of conventional lobular carcinoma, in a classic sense, while five others were a variant of this tumor. Individual tumor cells of lobular carcinoma were estimated to be well differentiated, both morphologically and functionally, revealing well developed intracytoplasmic organelles and a high percentage of alpha-lactalbumin content in the cytoplasm. Nevertheless, the tumor itself was characterized by a lack of any particular structural differentiation in the arrangement of cells. Based on the observation of the histologic features, invasive lobular carcinoma was subclassified into three groups, in situ predominant, intermediate, and diffuse infiltrating and with a definite correlation to the age of the patient and to the prognosis. Validity of this classification indicates that lobular carcinoma progresses gradually, even in the invasive phase, and can be categorized as a slowly growing subset of mammary carcinoma.