Neurotoxicity in rat cortical cells caused by N-methyl-D-aspartate (NMDA) and gp120 of HIV-1: induction and pharmacological intervention.

Incubation of highly enriched neurons from rat cerebral cortex with the human immunodeficiency virus type 1 (HIV-1) coat protein gp120 for 18 h results in fragmentation of DNA at internucleosomal linkers, a feature of apoptosis. We report that neurons respond to exposure to gp120 with an increased release of arachidonic acid via activation of phospholipase A2. This process is not inhibited by antagonists of the N-methyl-D-aspartate (NMDA) receptor channels. To investigate the influence of arachidonic acid on the sensitivity of NMDA receptor towards its against, low concentrations of NMDA were coadministered with arachidonic acid. Under these conditions the NMDA-mediated cytotoxicity was enhanced. We conclude that gp120 causes an activation of phospholipase A2, resulting in an increased release of arachidonic acid which in turn sensitizes the NMDA receptor. Two compounds were found to act cytoprotectively against the deleterious effect caused by gp120 on neurons: Memantine [1-amino-3,5-dimethyladamantane] and Flupirtine [2-amino-3-ethoxycarbonylamino-6-(4-fluoro-benzyl-amino)-pyridine maleate]. Both compounds have been found to display a potent cytoprotective effect on neurons treated with the excitatory amino acid NMDA or with the human immunodeficiency virus type 1 (HIV-1) coat protein gp120. The NMDA antagonist Memantine, a drug currently used in the therapy of spasticity and Parkinson's disease, prevented the effects of gp120 at micromolar concentrations. Flupirtine was previously found to be a centrally acting, nonopiate analgesic agent which additionally possesses anticonvulsant and muscle-relaxant activity at doses similar to those producing analgesia. The cytoprotective effect of Flupirtine in vitro was significant (above 10 microM). Considering the fact that both Memantine and Flupirtine display almost no clinical side effects, these drugs may prove useful both in preventing primary infection of brain cells with the HIV virus, as well as in treating the neurological disorders often associated with the immunodeficiency syndrome such as AIDS-related dementia.

The triaminopyridine flupirtine prevents cell death in rat cortical cells induced by N-methyl-D-aspartate and gp120 of HIV-1.

Flupirtine, a triaminopyridine derivative, is a non-opiate centrally acting analgesic agent with muscle relaxant properties. Now we show that this drug displays a potent cytoprotective effect on neurons (rat cortical cells) treated with (i) the excitatory amino acid N-methyl-D-aspartate (NMDA) or (ii) with the human immunodeficiency virus type 1 (HIV-1) coat protein gp120. In the absence of the drug the two agents cause a > 90% reduction of cell viability after a 18 h incubation. During this period the DNA in the cells undergoes fragmentation and shows a pattern which is typical for cell death. If the neurons were preincubated with flupirtine for 2 h and subsequently exposed to the cytotoxic agents an almost complete protection was achieved. The cytoprotective effect of flupirtine in vitro was significant (above 10 microM). Because flupirtine displays almost no clinical side effects and in light of the data presented here, flupirtine may be a promising drug also for the treatment of NMDA-mediated neurodegenerative disorders in general and for the treatment of AIDS-related encephalopathy in particular.

The role of wild-type and mutated N-ras in the malignant transformation of liver cells.

In order to determine the role of N-ras overexpression and mutation in malignant liver cell transformation, wild-type and mutated N-ras were transfected into the rat liver epithelial cell line OC/CDE 22, and N-ras expression, growth kinetics, growth in soft agar, and tumorigenicity in vivo as well as the involvement of the mitogen-activated protein kinase (MAPK) signal transduction pathway in the expression of the malignant phenotype were analyzed. Although OC/CDE 22 cells transfected with wild-type N-ras showed a high expression of N-ras at the mRNA and protein levels, the cells did not grow in soft agar and were not tumorigenic in vivo. In contrast, OC/CDE 22 cells transfected with mutated N-ras showed anchorage-independent growth and were tumorigenic. When cultured in fetal bovine serum-supplemented medium, OC/CDE 22 cells expressing mutant N-ras showed a higher proliferation rate than nontransfected OC/CDE 22 cells or OC/CDE 22 cells transfected with wild-type N-ras. When held in serum-free medium, untreated OC/CDE 22 cells did not grow at all, while OC/CDE 22 cells transfected with wild-type or mutant N-ras proliferated at a similar rate, which can be explained by the high MAPK activity in these cells. Selective inhibition of the MAPK cascade abolished the growth of OC/CDE 22 cells carrying mutant N-ras in soft agar; furthermore, these cells ceased pile up and formed monolayers on Petri dishes. Thus, activation of the MAPK signaling pathway, though alone not sufficient to malignantly transform liver cells (as shown in liver cells overexpressing wild-type N-ras), is not only essential for growth control but also for the expression of the malignant phenotype (as demonstrated in liver cells transformed by mutated N-ras).

The human p53 gene mutated at position 249 per se is not sufficient to immortalize human liver cells.

A particular point mutation of the tumor suppressor gene p53, namely a G-->T transversion at the third base of codon 249, is frequently detected in primary hepatocellular carcinomas from patients living in areas where the levels of dietary exposure to aflatoxin B1 and the rates of infection with the hepatitis B virus are very high. Very recently, a nontumorigenic liver epithelial cell line (HACL-1) with a finite life-span and expressing a number of hepatocyte-specific markers was established from a human hepatocellular adenoma in our laboratory. To analyze the role of mutated p53 in the immortalization of human liver cells, we transfected HACL-1 cells with an expression vector containing a human p53 complementary DNA mutated at the third base of codon 249 and analyzed the consequences of this gene transfer on the growth properties of this cell line. HACL-1 cells transfected with mutant p53 showed no increase in their life-span (when compared with HACL-1 cells transfected with the antibiotic resistance gene alone) and did not grow in soft agar, whereas transfection of wild-type p53 into HACL-1 cells led to a proliferation stop. Thus, these results strongly support the view that the mutation at codon 249 of the p53 gene may serve as a fingerprint for aflatoxin B1-induced hepatocellular carcinomas, but is not, by itself, sufficient to immortalize human liver cells.

Identification of frequent chromosomal aberrations in ductal adenocarcinoma of the pancreas by comparative genomic hybridization (CGH).

Despite the continuous progress in molecular methodology, the genetic events involved in the initiation and progression of ductal adenocarcinoma of the pancreas remain largely unknown. In this study, 33 pancreatic ductal adenocarcinomas were screened for genomic alterations by comparative genomic hybridization (CGH). To date, most CGH studies of pancreatic cancer have been based on cell lines. To emphasize genetic imbalances that are involved in the in vivo development and progression of pancreatic carcinoma only fresh-frozen or paraffin-embedded tumour samples were analysed in the present study. Twenty-two tumours (67%) showed genomic alterations involving up to three (12%) or more (55%) chromosomal regions. The number and nature of the genetic imbalances did not, however, correlate with tumour stage or grade. Chromosome 18 was preferentially altered in the tumours analysed. Frequent chromosomal losses were found at 18q, 10q, 8p, and 13q. Commonly gained regions were located on 8q and 3q. Moreover, high copy number amplifications of the chromosomal regions 5p, 8q22-ter, 12p12-cen, 19q12-13.2, and 20q were identified. These data provide evidence for the occurrence of characteristic genomic alterations which are of biological relevance for the genesis of pancreatic cancer. The identified altered chromosomal regions may harbour tumour genes which involved in the multistep process of pancreatic carcinogenesis.

c-MYC activation in primary and metastatic ductal adenocarcinoma of the pancreas: incidence, mechanisms, and clinical significance.

Amplification and overexpression of c-MYC is a common event in various neoplasias. Recently, comparative genomic hybridization (CGH) of primary pancreatic adenocarcinomas revealed a distinct high-level amplification of 8q23-qter, suggesting that c-MYC located on 8q24 may be a candidate oncogene. To evaluate the biological significance and prognostic value of c-MYC activation in pancreatic carcinoma, we performed interphase fluorescence in situ hybridization (FISH) and immunohistochemistry on a series of 69 primary pancreatic adenocarcinomas, 19 corresponding lymph node metastases, and 5 pancreatic intraductal lesions. Dual color FISH using a probe for c-MYC (8q24) and a centromeric probe for chromosome 8 revealed amplification of c-MYC in 32.3% and 29.4% of primary and metastatic tumors, respectively. Immunostaining identified c-MYC protein overexpression in 43.5% of primaries and 31.6% of metastases. Low concordance between positive FISH and immunostaining (13.4%) suggests multiple independent regulatory pathways of c-MYC activation. Statistical evaluation revealed significant correlation (alpha = 0.033) between c-MYC protein overexpression and histopathological tumor grade but absence of correlation with tumor stage or lymph node status. Analysis of pancreatic intraductal lesions showed c-MYC amplification and protein overexpression in two of five cases in which invasive carcinoma exhibited identical aberrations. We conclude that deregulation of c-MYC protein is common in pancreatic cancer and that it may be involved in early neoplastic development and progression rather than in locoregional spread of invasive cancer.

[Innovative cell culture methods in drug development]. / Möglichkeit der Nutzung von Zellkulturmethoden in der Arzneimittelentwicklung.

The animal studies necessary for drug registration are time-consuming, costly, and often stressful for the animals. Toxicological screening of drug candidates early in development with in vitro cell culture systems is therefore of relevance. In contrast to animal studies, in vitro cell culture methods are characterized by a low compound requirement and a short duration. Additionally it is possible to include mechanistic studies or to test for toxicity specific to humans. Therefore, early toxicological screening can provide a useful support for selecting the most promising drug candidate. Primary hepatocytes can be used to measure the cytotoxicity of a test compound. These results can be used to estimate general toxicity. Measuring endpoints like apoptosis, redox status, or gene expression profiles can help to answer mechanistic questions. The use of primary human hepatocytes provides early predictivity for hepatotoxicity specific to humans. Since teratogenic findings in animal studies often lead to abandonment of development, it is reasonable to use an in vitro embryotoxicity assay for early determination of the teratogenic potential of a compound, e.g. the embryonic stem cell test (EST) which was recently developed by ZEBET. In the EST embryonic stem cells are investigated for their preserved capability to differentiate into cardiomyocytes following drug exposure. In comparison cytotoxicity of the test substance is analyzed in embryonic stem cells and in differentiated fibroblast cells. In a validation study initiated by ECVAM the EST shows a high correlation with in vivo data.

Establishment and characterization of a nontumorigenic cell line derived from a human hepatocellular adenoma expressing hepatocyte-specific markers.

In the present study the establishment and characterization of a nontumorigenic liver epithelial cell line (HACL-1) derived from a human hepatocellular adenoma is described. The HACL-1 cells have a finite life span (i.e., they proliferate for a period of 2 months and then senesce), show cell-cell contact inhibition, do not grow in soft agar, are not tumorigenic when injected in nude mice, and possess a normal diploid karyotype. The cultured cells resemble hepatocytes, but exhibit some features of dedifferentiation. At the ultrastructural level the cells are endowed with round or oval nuclei, abundant cytoplasmic organelles, and varying amounts of glycogen. The rough endoplasmic reticulum is disorganized, while peroxisomes and matrix granules within mitochondria are lacking. HACL-1 cells are cytokeratin 18-positive as well as (transiently) albumin- and alpha-fetoprotein-positive, but do not express cytokeratin 19. Furthermore, no mutations were observed in exons 5-8 of the tumor suppressor gene p53. Taken together these results show that HACL-1 cells are nontumorigenic proliferating liver epithelial cells, which might prove to be of great value in future studies on diverse aspects of human liver cell biology and carcinogenesis.

Expression of MAGE antigens and analysis of the inflammatory T-cell infiltrate in human seminoma.

The MAGE gene family encodes antigens that are recognized by cytotoxic T-cells. The expression of MAGE antigens has been linked to tumor stage, and MAGE peptides are under investigation as possible vaccines. Seminomas are tumors that are typically accompanied by a heavy inflammatory infiltrate, but have not been studied with regard to their MAGE antigen expression and its correlation with the inflammatory infiltrate. We investigated, therefore, MAGE protein expression, the amount of cytotoxic T-cells, clonality of the lymphocytic infiltrate, apoptotic activity and occurrence of necrosis. Specimens of 27 patients with classical seminoma were examined by immunohistochemistry for CD4, CD8, CD56, CD45R0, beta2-microglobulin and HLA-DR. MAGE expression was detected with the monoclonal antibody 57B, reactive with MAGE-1, -3, -4, -6 and -12. Clonality of the inflammatory infiltrate was examined by multiplex polymerase chain reaction (PCR) analysis of the T-cell receptor rearrangement. Apoptotic cells were detected by DNA nick-end labeling of fragmented DNA, and the apoptotic index was determined semi-quantitatively. Expression of 57B was found in 19 (70%) of 27 seminomas. In all cases, more than 70% of T-cells expressed CD45R0. In four cases, a predominant infiltration of CD8-positive cytotoxic T-cells (CD4/CD8 ratio < 1) was present. However, 15 seminomas showed a CD4/CD8 ratio > 1. In all cases, infiltration of CD56-positive natural killer cells was only focal. HLA-DR expression was not detectable in tumor tissue; beta2-microglobulin was only focal in three cases. Analysis of the T-cell clonality revealed a polyclonal population. The apoptotic index was not significantly different in 57B-positive seminomas (4.15%) compared with 57B negative seminomas (3.80%). Also, no correlation between the 57B expression and the occurrence of necrosis was found. MAGE antigens are homogeneously expressed in most seminomas, but their presence does not appear to represent a dominant epitope responsible for the lymphocytic infiltrate.