Immortalized bovine pancreatic duct cells become tumorigenic after transfection with mutant k-ras.

Mutation of the K-ras gene is thought to be an early and important event in pancreatic carcinogenesis. In order to study the role of this molecular alteration in the transition from the normal to the neoplastic pancreatic cell, bovine pancreatic duct cells were first immortalized by SV40 large T antigen (Ag) complementary (c)DNA transfection and then transfected with a mutated K-ras gene. As did primary duct cells, the immortalized duct cells (more than 100 passages) expressed cytokeratins, carbonic anhydrase type-II, cystic fibrosis transmembrane conductance regulator (CFTR), and multidrug resistance (mdr). They grew as a single layer after transplantation under plastic domes and formed three-dimensional structures resembling ducts when grown on Matrigel. Cell growth was stimulated by insulin, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, but cells did not respond to gastrin and CCK-8. They did not form colonies in soft agar nor did they form tumors in nude mice. Immortalized cells transfected with mutated K-ras acquired the ability to form tumors after orthotopic injection into the nude mouse pancreas. It is concluded that SV 40 immortalized bovine pancreatic

The "point of no return" and the rate of progression in the natural history of IgA nephritis.

BACKGROUND: Based on the observation of 7 patients with chronic IgA nephritis and on a course to end-stage renal failure after several years, D'Amico et al. [1993] reported on a "point of no return" at 2.5 to 3 mg/dl serum creatinine. After exceeding this limit all 7 patients exhibited an irreversible progressive renal failure. PATIENTS AND METHODS: Therefore, 115 patients with IgA nephritis from the "German Glomerulonephritis Therapy Study" were examined in order to look for the existence of such a "point of no return". RESULTS: Three different courses could be distinguished: a stable chronic course with constantly normal or only minor elevated serum creatinine lasting for years (91 patients), a progressive course with continuously increasing serum creatinine (22 patients), and a rare (only 2 patients) early acute course with a short-term increase of serum creatinine followed by a rapid return to the normal range. After exceeding 3 mg/dl serum creatinine no remissions were observed in the progressive cases. Sixteen patients showed a rapid, continuously progressive course until end-stage renal failure with exactly the same progression as the 7 patients of D'Amico et al. Six patients of the 22 progressors were not observed long enough. The serum creatinine level doubled on average from 3 to 6 mg/dl within 10 months. CONCLUSION: Our study confirmed the existence of a "point of no return" at 3 mg/dl (265 micromol/l) during the natural course of chronic IgA nephritis.

Late occurrence of cysts in autosomal dominant medullary cystic kidney disease.

Medullary cystic kidney disease (MCD) is characterized by multiple renal cysts at the corticomedullary boundary area, by autosomal dominant inheritance, and by onset of chronic renal failure in the third decade of life. We report on a family with three affected individuals of both sexes in two generations presenting with end-stage renal failure at age 22-31 years. Primarily diagnoses considered included unclassified hereditary nephropathy and autosomal dominant polycystic kidney disease. Careful evaluation of all findings, initiated after investigation of renal morphology with CT, revealed features characteristic for MCD and led to the final diagnosis of MCD. We conclude that MCD is an important differential diagnosis for polycystic kidney disease in young adults with end-stage renal failure. Establishing the correct diagnosis has considerable impact for genetic counselling.

TGF-beta1-induced cell cycle arrest in renal mesangial cells involves inhibition of cyclin E-cdk 2 activation and retinoblastoma protein phosphorylation.

In glomerular disease, transforming growth factor-beta1 (TGF-beta1) has been demonstrated to exert anti-mitogenic and anti-inflammatory as well as fibrogenic effects. To better understand the TGF-beta1 action on glomerular cells at the molecular level, we investigated mechanisms of TGF-beta1-induced growth suppression in primary cultures of rat mesangial cells (MCs). TGF-beta1 (5 ng/ml) markedly inhibited proliferation of MCs incubated with PDGF, endothelin-1, bFGF, serotonin, or EGF, indicating that TGF-beta1 interferes with post-receptor signals of mitogenesis. TGF-beta1 did not affect mitogen-stimulated induction of the immediate early genes, c-fos, c-jun, and Egr-1 in MCs that occurred transiently at 30 to 120 minutes. Time-course studies revealed that TGF-beta1 inhibited DNA synthesis and MC replication when added up to six to eight hours after MC stimulation with PDGF. FACS analysis demonstrated that MCs had reached middle to late G1 phase of cell cycle progression at this timepoint. PDGF stimulation of MCs induced protein expression of the G1 phase cyclin D1 as well as the cyclin-dependent kinases cdk 4 and cdk 2. This was not significantly altered when MCs were coincubated with both, PDGF and TGF-beta1. However, TGF-beta1 prevented PDGF-elicited phosphorylation of the retinoblastoma tumor suppressor (pRb), a negative cell cycle regulator. Moreover, TGF-beta1 significantly reduced cyclin E-associated histone H1 kinase activity in the presence of PDGF. These results indicate that TGF-beta1 inhibits mitogen-stimulated MC growth by causing cell cycle arrest in late G1 phase. While TGF-beta1 does not alter the mitogen-induced expression and abundance of G1 phase cyclin D1 and cdks 4 and 2 in MCs, it inhibits cyclin E-cdk 2 activity, thus preventing mitogen-elicited phosphorylation and inactivation of pRb in G1 phase and transition to S phase.

Monogenetic hypertension and pheochromocytoma.

Hypertension attributable to pheochromocytoma is a very attractive model for the elucidation of the pathogenesis of hypertension. Sixteen different point mutations in the RET proto-oncogene and 30 mutations in the Von Hippel-Lindau (VHL) tumor suppressor gene have been identified so far associated with expression of pheochromocytoma. Each of these mutations initiates either the syndrome of multiple endocrine neoplasia type 2 (MEN 2) (MEN 2A and MEN 2B) or the VHL disease. Certain mutations in both genes are associated with the presence of pheochromocytoma. In general, these pheochromocytomas produce catecholamines that result in hypertension. Therefore, analysis for germline mutations in these genes are of practical value, because susceptibility to these diseases can be predicted in as yet clinically unaffected relatives.

Expression and function of receptors for extracellular matrix proteins in human ductal adenocarcinomas of the pancreas.

Extracellular matrix proteins (ECM) may influence cellular differentiation via their receptors, the integrins. We recently presented evidence that ductal adenocarcinomas of the pancreas are able to produce ECM in vitro and in vivo (Br J Cancer 1994;49:144-51). This study examines whether pancreatic carcinoma cells are able to interact with ECM by expressing functionally active integrins. In eight human pancreatic tumor cell lines (AsPC-1, BxPC-3, CAPAN-1 and -2, PANC, PaTu-2, -3, and -44) and six xenografted tumors RNA and protein expression of integrin subunits alpha 5 (fibronectin receptor), alpha 6 (laminin receptor), and alpha V (vitronectin receptor) was investigated. In addition, alpha 1-alpha 6 and alpha V as well as beta 1-beta 4 were studied by fluorescence-activated cell sorter analysis. Integrin function was tested by attachment assays. alpha 2, alpha 3, alpha 5, alpha 6, and alpha V as well as beta 1, beta 3, and beta 4 were expressed, at both the RNA and the protein level, by all pancreatic tumors in vitro and in vivo. The tumor cell lines showed dose dependent adhesion to collagen, fibronectin, and laminin. Integrin expression could be modulated in part by serum depletion. None of the tumors showed alpha 1, alpha 4, and beta 2. Tumor cell differentiation or production of ECM was not correlated with integrin expression. Pancreatic ductal adenocarcinomas express a certain pattern of functionally active integrins that enable interaction with most matrix proteins, e.g., collagen, fibronectin, and laminin. Pancreatic adenocarcinomas possess both the ligands and the receptors of the extracellular matrix network. We speculate that through both classes of molecules, the tumor cells may bind to fibroblasts and probably stimulate their growth. However, it remains unclear whether and how integrin-ECM interaction exerts an influence on tumor cell differentiation.

Lipoprotein(a) in patients with the nephrotic syndrome: influence of immunosuppression and proteinuria.

Lipoprotein(a) [Lp(a)] is a plasma lipoprotein whose structure and composition closely resemble that of low-density lipoproteins, but contains an additional protein called apolipoprotein(a) [apo(a)]. Factors which modulate plasma Lp(a) concentrations are poorly understood. The influence of nephrotic syndrome on Lp(a) levels was investigated in 103 patients with nephrotic syndrome: 72 with primary kidney disease and 31 with diabetic nephropathy. Nephrotic patients had significantly higher Lp(a) levels (mean 63 +/- 7 mg/dl; median 42 mg/dl) compared with controls (mean 22 +/- 2 mg/dl; median 8 mg/dl). Fifty-seven percent of the patients and 22% of the controls had values greater than 30 mg/dl. Within all apo(a) isoform classes, higher concentrations of Lp(a) were seen in the nephrotic patients compared with controls. In 17 patients with primary kidney disease remission of the nephrotic syndrome was induced by immunosuppressive treatment and Lp(a) concentration dropped in parallel with the reduction of proteinuria (pretreatment mean, 98 +/- 9 mg/dl vs. remission mean, 25 +/- 5 mg/dl). In 9 patients where multiple measurements were done, multiple regression analysis showed a strong relation of Lp(a) with the amount of proteinuria (p < 0.01). We conclude that most patients with the nephrotic syndrome have Lp(a) concentrations which are substantially elevated compared with control subjects of the same apo(a) isoform. Because Lp(a) concentrations are substantially reduced when remission of the nephrotic syndrome is induced by immunosuppressive drugs, it is likely that nephrotic syndrome directly results in elevation of Lp(a). The high levels of Lp(a) in nephrotic syndrome could potentially cause glomerular injury as well as increase the risk of atherosclerosis and thrombotic events associated with this disorder.

The German Glomerulonephritis Therapy Study: 10 years of controlled randomized trials for the treatment of idiopathic glomerulonephritis.

The German Collaborative Glomerulonephritis Therapy Study, which celebrated its 10th anniversary in 1996, has collected data on more than 1,000 patients with biopsy-proven glomerulonephritis. 929 patients could be evaluated and 500 were treated according to at least one of various protocols developed for a randomized controlled trial. Current results show that prednisolone is effective in minimal-change nephropathy, and in combination with other immunosuppressants it can reduce proteinuria in individual cases of focal and segmental glomerulosclerosis, membranous glomerulonephritis and nephrotic IgA nephropathy. The majority of tested treatment protocols did not prove to be superior to symptomatic therapy for long-term outcome.

Consequences of direct genetic testing for germline mutations in the clinical management of families with multiple endocrine neoplasia, type II.

OBJECTIVE: Multiple endocrine neoplasia, type II (MEN-II) is an autosomal dominant disorder characterized by tumors of thyroid C cells and pheochromocytoma. Recently, germline mutations in the RET proto-oncogene have been identified in patients with MEN-II. The aims of this study were (1) to define the mutations in clinically diagnosed MEN-II families, (2) to compare the results of genetic and biochemical testing, and (3) to evaluate the impact of mutation analyses for the members of these families. DESIGN: Register-based survey study of clinically affected and unaffected members of MEN-II families. SETTING: Register of families from Germany and Spain with pheochromocytomas. Two research laboratories at Cambridge University in the United Kingdom. PATIENTS: We investigated consenting affected and unaffected members belonging to a series of 10 families who met the clinical criteria for MEN-II. MAIN OUTCOME MEASURES: (1) Presence or absence of germline mutation in the RET proto-oncogene in affected and unaffected members of the 10 families, and (2) in the absence of RET mutation in a given family, presence or absence of germline mutation in the von Hippel-Lindau (VHL) gene, which is the susceptibility gene involved in a closely related syndrome, von Hippel-Lindau disease. RESULTS: In eight of these families, RET mutations were identified. The specific mutations were detected in all affected members. The remaining two families without RET mutations were subsequently shown to have a mutation within the VHL gene. The VHL mutations were identified in both families and represent a previously undescribed base change. After identification of the mutation, premorbid genetic testing was performed in all MEN-II and VHL families, resulting in detection of asymptomatic carriers in the MEN-II families. Clinically, the two VHL families differed from the eight MEN-II families by the presence of a C-cell tumor in only one individual from each family and extra-adrenal pheochromocytoma in three of nine affected individuals in the two families combined. CONCLUSIONS: The diagnosis of MEN-II should be confirmed by molecular genetic analysis and the diagnosis of VHL syndrome should be considered for families with an absence of RET mutations and a preponderance of pheochromocytomas.

Effect of aspirin and dipyridamole on proteinuria in idiopathic membranoproliferative glomerulonephritis: a multicentre prospective clinical trial. Collaborative Glomerulonephritis Therapy Study Group (CGTS)

Idiopathic membranoproliferative glomerulonephritis (MPGN) has a poor prognosis, with 90% of patients requiring dialysis treatment after 20 years regardless of therapy. Up to 34% of patients may die due to thrombotic complications or sepsis. This study investigates the influence of aspirin plus dipyridamole on proteinuria and renal function in nephrotic MPGN patients with moderately reduced glomerular filtration rate. Eighteen patients with biopsy-proven MPGN (15 type I, 3 type II) and nephrotic syndrome were randomly assigned to receive protein restriction, antihypertensive therapy (control group) or in addition aspirin and dipyridamole (treatment group). Patients were prospectively followed for a mean of 36 months. Serum creatinine remained unchanged after 36 months compared to baseline in both groups. In the treatment group proteinuria was reduced from 8.3 +/- 1.4 to 1.6 +/- 0.7 g/day (P < 0.05). In control patients proteinuria decreased from 7.1 +/- 1.6 to 4.3 +/- 1.1 g/day. After 36 months proteinuria was significantly lower in the treatment group compared to control (P < 0.02 Mann-Whitney rank sum test). In conclusion, aspirin plus dipyridamole may be of value in reversing nephrotic syndrome and associated risks in patients with MPGN and moderately reduced renal function.

Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins.

Pancreatic ductal adenocarcinomas are characterised by a dense connective tissue reaction. To test the hypothesis that stroma components are synthesised and produced by the tumour cells themselves, eight cell lines as well as six xenografted tumours from human ductal adenocarcinomas of the pancreas were examined for the expression of extracellular matrix proteins (ECM), using cDNA probes and antibodies to collagen types I, III and IV, vitronectin, fibronectin, undulin and laminin. All tumour cell lines (CAPAN-1, CAPAN-2, AsPC-1, BxPC-3, PANC-1, PaCa-2, PaCa-3, PaCa-44) and xenografted human pancreatic tumours expressed at least one of the examined ECM at the RNA (collagen type IV > laminin = fibronectin = vitronectin > collagen type III > undulin > collagen type I) or protein level (collagen type IV = collagen type III > vitronectin > laminin > collagen type I = fibronectin > undulin). In nude mouse tumours expression of laminin and collagen I was most pronounced in well-differentiated carcinomas. In a few tumours, collagen type III, vitronectin and undulin were expressed on the luminal side of the neoplastic glands, suggesting loss of normal polar differentiation. Incubation with fetal calf serum modulated ECM RNA levels to a varying extent in all but one cell line (AsPC-1). The results suggest that human pancreatic ductal adenocarcinomas cells are capable of synthesising and producing extracellular matrix proteins in vitro and in vivo, but that the extent and pattern of ECM expression differs between the various tumours and conditions tested.

[Pheochromocytoma as dominant manifestation of v. Hippel-Lindau syndrome]. / Phäochromozytom als dominierende Manifestation des v. Hippel-Lindau-Syndroms.

A diagnosis of von Hippel-Lindau syndrome was made in two families originating from the same part of the Black Forest but apparently unrelated. Nine affected persons (seven males and two females) had a total of 17 tumours: retinal angioma (4), haemangioblastoma of the CNS (1), and phaeochromocytoma (12). Three of the affected persons and eight of the tumours (six phaeochromocytomas, two retinal angiomas) were diagnosed by family screening. Phaeochromocytoma was diagnosed in eight persons; in four it was the only symptomatic lesion. After extensive diagnostic tests the phaeochromocytoma was the sole tumour in four. Despite severe symptoms the diagnosis of von Hippel-Lindau syndrome had not been made prior to the screening examinations because either the common aetiology of the tumour was not known or there was insufficient exchange of information between the two families.--It is recommended that in each case of phaeochromocytoma von Hippel-Lindau syndrome should be excluded so that lesions can be discovered early in other organs and in other affected family members. If the syndrome is present, annual examinations are indicated because of asynchronous and multi-focal tumour growth.