Cathepsin-B and cathepsin-L expression levels do not correlate with sensitivity of tumour cells to TNF-alpha-mediated apoptosis.

Recently, evidence has been accumulated that besides the caspase proteases, lysosomal cathepsins may play a role in apoptosis induction. This is especially significant as many human tumour cells express high levels of cathepsins, which might sensitise these cells to specific proapoptotic stimuli mediated by cathepsins. We found that TNF-alpha-mediated DNA fragmentation in tumour cells was significantly reduced in the presence of E64d and CA074Me, two inhibitors of lysosomal cysteine proteases. Transient transfection of cathepsin-B (Cath-B) and -L (Cath-L) resulting in expression levels comparable to those found in many tumours did not sensitise tumour cells to TNF-alpha-mediated apoptosis. As lysosomal proteases are thought to be activated by their release from this organelle into the cytosol, we used the lysosomotropic detergent N-dodecyl-imidazole-HCl (NDI-HCl) to disturb lysosomal integrity efficiently and trigger the release of its proteolytic content into the cytosol. Treatment of HeLa cells with NDI-HCl resulted in cell death, which, however, could also not be influenced by augmented Cath-B or -L expression levels. Therefore, our data do not support the hypothesis that the high Cath-B or -L expression levels frequently detected in tumour cells might be exploited to target selectively those tumours for an enhanced cell death effect induced by lysosomotropic agents.

Ceramide induces cell death in the human prostatic carcinoma cell lines PC3 and DU145 but does not seem to be involved in Fas-mediated apoptosis.

Treatment of cells with synthetic C2-ceramide has been reported to induce apoptosis in several cell systems, and endogenously formed ceramide has been proposed to act as a second messenger, activating signaling pathways which contribute to the execution of apoptotic cell death after Fas ligation or tumor necrosis factor receptor-1 ligation. In this study, we examined the effect of exogenously administered C2-ceramide on the human prostatic carcinoma cell lines PC3 (Fas-sensitive) and DU145 (Fas-resistant). In both cell lines, C2-ceramide induced cell death in a dose-dependent manner, whereas a structural analog, C2-dihydroceramide, did not. The pan-caspase inhibitor zVAD-fmk did not prevent C2-ceramide-induced cell death but did prevent C2-ceramide-induced DNA fragmentation, indicating that apoptotic and non-apoptotic mechanisms are involved in C2-ceramide-induced death. Interestingly, cycloheximide prevented C2-ceramide-induced DNA fragmentation, indicating that ceramide-induced apoptosis in PC3 and DU145 requires new protein synthesis. In addition, because cycloheximide converts Fas-resistant DU145 to Fas-sensitive as assessed by DNA fragmentation, ceramide does not seem to play a major role in the Fas-mediated pathway in this cell line. We also determined the levels of endogenous sphingomyelin after Fas ligation in PC3. No decrease of sphingomyelin levels could be detected after Fas activation. We conclude that sphingomyelinase-generated ceramide does not play a role in Fas-mediated apoptosis in PC3, and that there are fundamental differences in the mechanisms of cell death induced by C2-ceramide and Fas ligation.

p53 is involved in tumor necrosis factor-alpha-induced apoptosis in the human prostatic carcinoma cell line LNCaP.

The human prostatic carcinoma cell line LNCaP is sensitive to TNF-alpha treatment and expresses wild-type p53. To analyse the possible role of p53 in TNF-alpha-mediated apoptosis, we generated a derivative of LNCaP, LN-56, expressing a dominant-negative element of p53, GSE56. P53 inactivation in LN-56 was associated with an increased resistance to apoptosis induced by TNF-alpha. Surface expression of TNF-alpha receptors was unchanged in LN-56 compared to LNCaP. TNF-alpha treatment resulted in accumulation of p53 in LNCaP and upregulation of p21/WAF1. Activation of caspase-7 and PARP proteolysis were delayed in LN-56 under TNF-alpha treatment. TNF-alpha-induced apoptosis in LNCaP cells was accompanied by caspase-dependent proteolysis of p21/WAF1 and Rb, which was significantly attenuated in LN-56. Cytochrome c release was induced by TNF-alpha treatment in both cell lines, but caspase-9 was not activated. LNCaP and LN-56 were injected s.c. in nude mice and tumors were identified in all LN-56, but not LNCaP, bearing mice indicating that p53 plays an important role in growth control of prostatic neoplasms. Interestingly, accumulation of p53 in TNF-alpha-treated LNCaP cells was decreased in the presence of the caspase inhibitor Z-VAD-FMK, suggesting a new role of activated caspases in acceleration of p53 response. In summary, these results indicate that p53 is involved in TNF-alpha-mediated apoptosis in LNCaP.

Cytochrome c is involved in Fas-mediated apoptosis of prostatic carcinoma cell lines.

We have shown previously that the pathways leading to Fas-mediated apoptosis in prostatic carcinoma cell lines are intact, because apoptosis can be triggered either by Fas ligation alone in the Fas-sensitive cell lines PC3 and ALVA31 or by rendering the Fas-resistant cell lines DU145 and JCA1 Fas-sensitive by combined treatment with anti-Fas monoclonal antibody and cycloheximide (O. W. Rokhlin et al., Cancer Res., 57: 1758-1768, 1997). In this study, we demonstrate that two of the early events after Fas ligation are the release of cytochrome c from the mitochondria and activation of caspase-9. We also found that Bid is processed after Fas ligation and thus might activate the mitochondria-dependent apoptotic cascade. In a cell-free system, cytochrome c induced caspase-3-like activity in cytoplasmic extracts from all four cell lines studied, although differences in the level of enzymatic activity were observed. Western blot analysis revealed that caspase-7 is activated by cytochrome c at the same level in all extracts, whereas expression and activation of caspase-3 varied considerably. Cytochrome c-activated extracts displayed different abilities in the induction of apoptotic features in isolated nuclei such as morphological changes and DNA fragmentation. However, differences in nuclear apoptotic activity induced by cytochrome c did not correlate with the level of caspase-3 like activity in the different extracts. These results suggest that the mitochondrial pathway is involved in Fas-mediated apoptosis in prostatic carcinoma cell lines and that, in addition to caspase-7 and caspase-3, there are other factors that confer nuclear apoptotic activity.

Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain.

More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered novel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for PTT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study, <20% represent C-->T or G-->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C-->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation rate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region, with major roles played by exons 12a and 15 and part of exon 16.

Analysis of the NF1 gene by temperature gradient gel electrophoresis reveals a high incidence of mutations in exon 4b.

A total of 196 unrelated patients with neurofibromatosis type 1 (NF1) was screened for mutations in exons 4a-c of the NF1 gene by temperature gradient gel electrophoresis (TGGE) of polymerase chain reaction (PCR)-amplified genomic DNA fragments using intron-based primers. DNA samples with abnormal TGGE band patterns were subjected to sequence analysis. Sequence alterations were identified in ten patients (5.1%): 496delGT (1), 499delTGTT (4), T528A = D176E (2), T539A = L180X (1), 540insA (1), C574T = R192X (1). Thus, a total of six different mutations was identified in exon 4b but none in exons 4a and 4c. Only the missense mutation D176E, which we assume to be a nonpathogenic polymorphism, and the 4-base pair (bp) deletion 499delTGTT have been described before. The reason for the high incidence of mutations in exon 4b is obviously a tetranucleotide tandem repeat comprising nucleotides 495-502 (TGTTTGTT) that may give rise to slipped mispairing and subsequent deletion of one repeat unit during replication. Additionally, the recurrent 4 bp deletion was found as a second hit in a malignant schwannoma of a further NF1 patient, suggesting that microlesions may be as frequent among somatic as among germline mutations. This is the first report of a systematic study of NF1 exons 4a-c in a large group of NF1 patients.

Recurrent NF1 gene mutation in a patient with oligosymptomatic neurofibromatosis type 1 (NF1).

We report a 21-year-old male with symptomatic optic glioma who does not fulfill the diagnosis of neurofibromatosis 1 (NF1) according to standard NIH criteria. Analysis of the NF1 gene revealed a recurrent mutation in exon 37 (C6792A or Y2264X). This nonsense mutation causes skipping of exon 37 during the splicing process and is predicted to result in a protein shortened by 34 amino acid residues. The mutation was detected in all tissues examined (blood lymphocytes, oral mucosa, and dermal fibroblasts). The same mutation was previously found in 3 patients with clinically confirmed NF1. To our knowledge, this is the first report of an adult patient carrying a putative (non-mosaic) NF1 gene mutation in multiple tissues but not fulfilling the NIH criteria for the clinical diagnosis of NF1.

McCune-Albright syndrome: clinical and molecular evidence of mosaicism in an unusual giant patient.

Molecular genetics recently uncovered the mystery of the protean picture of McCune-Albright syndrome by identification of the somatic gain of function mutations in the GNAS1 gene. Here we present an adult patient with fibrous dysplasia and an endocrinopathy resulting in unusual giant height. The clinical diagnosis in the patient could be confirmed by molecular investigations in tissues involved in the process of fibrous dysplasia.

Selective disactivation of neurofibromin GAP activity in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a common familial tumour syndrome with multiple clinical features such as neurofibromas, café-au-lait spots (CLS), iris Lisch nodules, axillary freckling, optic glioma, specific bone lesions and an increased risk of malignant tumours. It is caused by a wide spectrum of mutations affecting the NF1 gene. Most mutations result in the loss of one allele at the DNA, mRNA or protein level and thus in the loss of any function of the gene product neurofibromin. The idea of the simultaneous loss of several different neurofibromin functions has been postulated to explain the pleiotropic effects of its loss. However, we have identified a novel missense mutation in a family with a classical multi-symptomatic NF1 phenotype, including a malignant schwannoma, that specifically abolishes the Ras-GTPase-activating function of neurofibromin. In this family, Arg1276 had mutated into proline. Based on complex biochemical studies as well as the analysis of the crystal structure of the GTPase-activating protein (GAP) domain of p120GAP in the presence of Ras, we unequivocally identified this amino acid as the arginine finger of the neurofibromin GAP-related domain (GRD)-the most essential catalytic element for RasGAP activity. Here, we present data demonstrating that the mutation R1276P, unlike previously reported missense mutations of the GRD region, does not impair the secondary and tertiary protein structure. It neither reduces the level of cellular neurofibromin nor influences its binding to Ras substantially, but it does completely disable GAP activity. Our findings provide direct evidence that failure of neurofibromin GAP activity is the critical element of NF1 pathogenesis. Thus, therapeutic approaches aimed at the reduction of Ras.GTP levels in neural crest-derived cells can be expected to relieve most of the NF1 symptoms.

Two novel mutations in exons 19a and 20 and a BsaBI [correction of BsaI] polymorphism in a newly characterized intron of the neurofibromatosis type 1 gene.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. It is caused by mutations in the NF1 gene, which comprises 60 exons and is located on chromosome 17q11.2. A total of 170 unrelated NF1 patients were screened for mutations in four exons by temperature-gradient gel electrophoresis. Preparatory work revealed the presence of a previously uncharacterized intron (19a) in what was previously designated exon 19; this allowed us to develop assays for genomic mutation screening in the newly defined exons 19a and 19b. Two novel NF1 mutations were detected: a single-base insertion in exon 19a creating a frameshift, and a second mutation affecting the splice donor site of intron 20 and leading to skipping of exon 20. A novel BsaBI polymorphism was identified in intron 19a.