A Fas-associated protein factor, FAF1, potentiates Fas-mediated apoptosis.

Fas, a member of the tumor necrosis factor receptor family, can induce apoptosis when activated by Fas ligand binding or anti-Fas antibody crosslinking. Genetic studies have shown that a defect in Fas-mediated apoptosis resulted in abnormal development and function of the immune system in mice. A point mutation in the cytoplasmic domain of Fas (a single base change from T to A at base 786), replacing isoleucine with asparagine, abolishes the signal transducing property of Fas. Mice homozygous for this mutant allele (lprcg/lprcg mice) develop lymphadenopathy and a lupus-like autoimmune disease. Little is known about the mechanism of signal transduction in Fas-mediated apoptosis. In this study, we used the two-hybrid screen in yeast to isolate a Fas-associated protein factor, FAF1, which specifically interacts with the cytoplasmic domain of wild-type Fas but not the lprcg-mutated Fas protein. This interaction occurs not only in yeast but also in mammalian cells. When transiently expressed in L cells, FAF1 potentiated Fas-induced apoptosis. A search of available DNA and protein sequence data banks did not reveal significant homology between FAF1 and known proteins. Therefore, FAF1 is an unusual protein that binds to the wild type but not the inactive point mutant of Fas. FAF1 potentiates Fas-induced cell killing and is a candidate signal transducing molecule in the regulation of apoptosis.

Two subsites in the binding domain of the acetylcholine receptor: an aromatic subsite and a proline subsite.

The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is localized in the alpha-subunit within a domain containing the tandem Cys-192 and -193. By analyzing the binding-site region of AcChoR from animal species that are resistant to alpha-neurotoxins, we have previously shown that four residues in this region, at positions 187, 189, 194, and 197, differ between animals sensitive (e.g., mouse) and resistant (e.g., mongoose and snake) to alpha-bungarotoxin (alpha-BTX). In the present study, we performed site-directed mutagenesis on a fragment of the mongoose AcChoR alpha-subunit (residues 122-205) and exchanged residues 187, 189, 194, and 197, either alone or in combination, with those present in the mouse alpha-subunit sequence. Only the mongoose fragment in which all four residues were mutated to the mouse ones exhibited alpha-BTX binding similar to that of the mouse fragment. The mongoose double mutation in which Leu-194 and His-197 were replaced with proline residues, which are present at these positions in the mouse AcChoR and in all other toxin binders, bound alpha-BTX to approximately 60% of the level of binding exhibited by the mouse fragment. In addition, replacement of either Pro-194 or -197 in the mouse fragment with serine and histidine, respectively, markedly decreased alpha-BTX binding. All other mutations resulted in no or just a small increase in alpha-BTX binding. These results have led us to propose two subsites in the binding domain for alpha-BTX: the proline subsite, which includes Pro-194 and -197 and is critical for alpha-BTX binding, and the aromatic subsite, which includes amino acid residues 187 and 189 and determines the extent of alpha-BTX binding.

Mutagenesis of the human apolipoprotein B gene in a yeast artificial chromosome reveals the site of attachment for apolipoprotein(a).

Lipoprotein(a) [Lp(a)] is a lipoprotein formed by the disulfide linkage of apolipoprotein (apo) B100 of a low density lipoprotein particle to apolipoprotein(a). Prior studies have suggested that one of the C-terminal Cys residues of apo-B100 is involved in the disulfide linkage of apo-B100 to apo(a). To identify the apo-B100 Cys residue involved in the formation of Lp(a), we constructed a yeast artificial chromosome (YAC) spanning the human apo-B gene and used gene-targeting techniques to change Cys-4326 to Gly. The mutated YAC DNA was used to generate transgenic mice expressing the mutant human apo-B100 (Cys4326Gly). Unlike the wild-type human apo-B100, the mutant human apo-B100 completely lacked the ability to bind to apo(a) and form Lp(a). This study demonstrates that apo-B100 Cys-4326 is required for the assembly of Lp(a) and shows that gene targeting in YACs, followed by the generation of transgenic mice, is a useful approach for analyzing the structure of large proteins coded for by large genes.

Mutations in the MSH3 gene preferentially lead to deletions within tracts of simple repetitive DNA in Saccharomyces cerevisiae.

Eukaryotic genomes contain tracts of DNA in which a single base or a small number of bases are repeated (microsatellites). Mutations in the yeast DNA mismatch repair genes MSH2, PMS1, and MLH1 increase the frequency of mutations for normal DNA sequences and destabilize microsatellites. Mutations of human homologs of MSH2, PMS1, and MLH1 also cause microsatellite instability and result in certain types of cancer. We find that a mutation in the yeast gene MSH3 that does not substantially affect the rate of spontaneous mutations at several loci increases microsatellite instability about 40-fold, preferentially causing deletions. We suggest that MSH3 has different substrate specificities than the other mismatch repair proteins and that the human MSH3 homolog (MRP1) may be mutated in some tumors with microsatellite instability.

Cryptorchidism and homeotic transformations of spinal nerves and vertebrae in Hoxa-10 mutant mice.

Homozygous mice mutated by homologous recombination for the AbdB-related Hoxa-10 gene are viable but display homeotic transformations of vertebrae and lumbar spinal nerves. Mutant males exhibit unilateral or bilateral criptorchidism due to developmental abnormalities of the gubernaculum, resulting in abnormal spermatogenesis and sterility. These results reveal an important role of Hoxa-10 in patterning posterior body regions and suggest that Hox genes are involved in specifying regional identity of both segmented and nonovertly segmented structures of the developing body.

Role of glycogen synthase kinase 3 beta as a negative regulator of dorsoventral axis formation in Xenopus embryos.

The dorsoventral axis is established early in Xenopus development and may involve signaling by Wnts, a family of Wnt1-protooncogene-related proteins. The protein kinase shaggy functions in the wingless/Wnt signaling pathway, which operates during Drosophila development. To assess the role of a closely related kinase, glycogen synthase kinase 3 beta (GSK-3 beta), in vertebrate embryogenesis, we cloned a cDNA encoding a Xenopus homolog of GSK-3 beta (XGSK-3 beta). XGSK-3 beta-specific transcripts were detected by Northern analysis in Xenopus eggs and early embryos. Microinjection of the mRNA encoding a catalytically inactive form of rat GSK-3 beta into a ventrovegetal blastomere of eight-cell embryos caused ectopic formation of a secondary body axis containing a complete set of dorsal and anterior structures. Furthermore, in isolated ectodermal explants, the mutant GSK-3 beta mRNA activated the expression of neural tissue markers. Wild-type XGSK-3 beta mRNA suppressed the dorsalizing effects of both the mutated GSK-3 beta and Xenopus dishevelled, a proposed upstream signaling component of the same pathway. These results strongly suggest that XGSK-3 beta functions to inhibit dorsoventral axis formation in the embryo and provide evidence for conservation of the Wnt signaling pathway in Drosophila and vertebrates.

Constitutive phosphorylation of I kappa B alpha by casein kinase II.

The NF-kappa B/Rel proteins are sequestered in the cytoplasm in association with the phosphorylated form of I kappa B alpha. Upon induction with a wide variety of agents, the activity of NF-kappa B/Rel proteins is preceded by the rapid degradation of I kappa B alpha protein. We report the identification and partial purification of a cellular kinase from unstimulated or stimulated murine cells, which specifically phosphorylates the C terminus of I kappa B alpha. There are several consensus sites for casein kinase II (CKII) in the C-terminal region of I kappa B alpha. Additionally, the activity of the cellular kinase is blocked by antibodies against the alpha subunit of CKII. No phosphorylation of the C-terminal region of I kappa B alpha can be detected if the five possible serine and threonine residues that can be phosphorylated by CKII are mutated to alanine. A two-dimensional tryptic phosphopeptide map of I kappa B alpha from unstimulated cells was identical to that obtained by in vitro phosphorylation of I kappa B alpha with the partially purified cellular kinase. We propose that constitutive phosphorylation of I kappa B alpha is carried out by CKII.

Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice.

Surfactant protein B (SP-B) is an 8.7-kDa, hydrophobic protein that enhances the spreading and stability of surfactant phospholipids in the alveolus. To further assess the role of SP-B in lung function, the SP-B gene was disrupted by homologous recombination in murine mouse embryonic stem cells. Mice with a single mutated SP-B allele (+/-) were unaffected, whereas homozygous SP-B -/- offspring died of respiratory failure immediately after birth. Lungs of SP-B -/- mice developed normally but remained atelectatic in spite of postnatal respiratory efforts. SP-B protein and mRNA were undetectable and tubular myelin figures were lacking in SP-B -/- mice. Type II cells of SP-B -/- mice contained no fully formed lamellar bodies. While the abundance of SP-A and SP-C mRNAs was not altered, an aberrant form of pro-SP-C, 8.5 kDa, was detected, and fully processed SP-C peptide was markedly decreased in lung homogenates of SP-B -/- mice. Ablation of the SP-B gene disrupts the routing, storage, and function of surfactant phospholipids and proteins, causing respiratory failure at birth.

The product of the ataxia-telangiectasia group D complementing gene, ATDC, interacts with a protein kinase C substrate and inhibitor.

Ataxia-telangiectasia (AT) is an autosomal recessive human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from individuals with AT show sensitivity to ionizing radiation, elevated recombination, cell cycle abnormalities, and aberrant cytoskeletal organization. The molecular basis of the defect is unknown. A candidate AT gene (ATDC) was isolated on the basis of its ability to complement the ionizing radiation sensitivity of AT group D fibroblasts. Whether ATDC is mutated in any AT patients is not known. We have found that the ATDC protein physically interacts with the intermediate-filament protein vimentin, which is a protein kinase C substrate and colocalizing protein, and with an inhibitor of protein kinase C, hPKCI-1. Indirect immunofluorescence analysis of cultured cells transfected with a plasmid encoding an epitope-tagged ATDC protein localizes the protein to vimentin filaments. We suggest that the ATDC and hPKCI-1 proteins may be components of a signal transduction pathway that is induced by ionizing radiation and mediated by protein kinase C.

The human papilloma virus 16E6 gene sensitizes human mammary epithelial cells to apoptosis induced by DNA damage.

Programmed cell death (apoptosis) is a normal physiological process, which could in principle be manipulated to play an important role in cancer therapy. The key importance of p53 expression in the apoptotic response to DNA-damaging agents has been stressed because mutant or deleted p53 is so common in most kinds of cancer. An important strategy, therefore, is to find ways to induce apoptosis in the absence of wild-type p53. In this paper, we compare apoptosis in normal human mammary epithelial cells, in cells immortalized with human papilloma virus (HPV), and in mammary carcinoma cell lines expressing wild-type p53, mutant p53, or no p53 protein. Apoptosis was induced with mitomycin C (MMC), a DNA cross-linking and damaging agent, or with staurosporine (SSP), a protein kinase inhibitor. The normal and HPV-transfected cells responded more strongly to SSP than did the tumor cells. After exposure to MMC, cells expressing wild-type p53 underwent extensive apoptosis, whereas cells carrying mutated p53 responded weakly. Primary breast cancer cell lines null for p53 protein were resistant to MMC. In contrast, two HPV immortalized cell lines in which p53 protein was destroyed by E6-modulated ubiquitinylation were highly sensitive to apoptosis induced by MMC. Neither p53 mRNA nor protein was induced in the HPV immortalized cells after MMC treatment, although p53 protein was elevated by MMC in cells with wild-type p53. Importantly, MMC induced p21 mRNA but not p21 protein expression in the HPV immortalized cells. Thus, HPV 16E6 can sensitize mammary epithelial cells to MMC-induced apoptosis via a p53- and p21-independent pathway. We propose that the HPV 16E6 protein modulates ubiquitin-mediated degradation not only of p53 but also of p21 and perhaps other proteins involved in apoptosis.

A human RNase E-like activity that cleaves RNA sequences involved in mRNA stability control.

We have detected an endoribonucleolytic activity in human cell extracts that processes the Escherichia coli 9S RNA and outer membrane protein A (ompA) mRNA with the same specificity as RNase E from E. coli. The human enzyme was partially purified by ion-exchange chromatography, and the active fractions contained a protein that was detected with antibodies shown to recognize E. coli RNase E. RNA containing four repeats of the destabilizing motif AUUUA and RNA from the 3' untranslated region of human c-myc mRNA were also found to be cleaved by E. coli RNase E and its human counterpart in a fashion that may suggest a role of this activity in mammalian mRNA decay. It was also found that RNA containing more than one AUUUA motif was cleaved more efficiently than RNA with only one or a mutated motif. This finding of a eukaryotic endoribonucleolytic activity corresponding to RNase E indicates an evolutionary conservation of the components of mRNA degradation systems.

Identification of the human prostatic carcinoma oncogene PTI-1 by rapid expression cloning and differential RNA display.

Elucidating the relevant genomic changes mediating development and evolution of prostate cancer is paramount for effective diagnosis and therapy. A putative dominant-acting nude mouse prostatic carcinoma tumor-inducing gene, PTI-1, has been cloned that is expressed in patient-derived human prostatic carcinomas but not in benign prostatic hypertrophy or normal prostate tissue. PTI-1 was detected by cotransfecting human prostate carcinoma DNA into CREF-Trans 6 cells, inducing tumors in nude mice, and isolating genes displaying increased expression in tumor-derived cells by using differential RNA display (DD). Screening a human prostatic carcinoma (LNCaP) cDNA library with a 214-bp DNA fragment found by DD permitted the cloning of a full-length 2.0-kb PTI-1 cDNA. Sequence analysis indicates that PTI-1 is a gene containing a 630-bp 5' sequence and a 3' sequence homologous to a truncated and mutated form of human elongation factor 1 alpha. In vitro translation demonstrates that the PTI-1 cDNA encodes a predominant approximately 46-kDa protein. Probing Northern blots with a DNA fragment corresponding to the 5' region of PTI-1 identifies multiple PTI-1 transcripts in RNAs from human carcinoma cell lines derived from the prostate, lung, breast, and colon. In contrast, PTI-1 RNA is not detected in human melanoma, neuroblastoma, osteosarcoma, normal cerebellum, or glioblastoma multiforme cell lines. By using a pair of primers recognizing a 280-bp region within the 630-bp 5' PTI-1 sequence, reverse transcription-PCR detects PTI-1 expression in patient-derived prostate carcinomas but not in normal prostate or benign hypertrophic prostate tissue. In contrast, reverse transcription-PCR detects prostate-specific antigen expression in all of the prostate tissues. These results indicate that PTI-1 may be a member of a class of oncogenes that could affect protein translation and contribute to carcinoma development in human prostate and other tissues. The approaches used, rapid expression cloning with the CREF-Trans 6 system and the DD strategy, should prove widely applicable for identifying and cloning additional human oncogenes.

Premature suture closure and ectopic cranial bone in mice expressing Msx2 transgenes in the developing skull.

The coordinate growth of the brain and skull is achieved through a series of interactions between the developing brain, the growing bones of the skull, and the fibrous joints, or sutures, that unite the bones. These interactions couple the expansion of the brain to the growth of the bony plates at the sutures. Craniosynostosis, the premature fusion of the bones of the skull, is a common birth defect (1 in 3000 live births) that disrupts coordinate growth and often results in profoundly abnormal skull shape. Individuals affected with Boston-type craniosynostosis, an autosomal dominant disorder, bear a mutated copy of MSX2, a homeobox gene thought to function in tissue interactions. Here we show that expression of the mouse counterpart of this mutant gene in the developing skulls of transgenic mice causes craniosynostosis and ectopic cranial bone. These mice provide a transgenic model of craniosynostosis as well as a point of entry into the molecular mechanisms that coordinate the growth of the brain and skull.

Effective ribozyme delivery in plant cells.

Hammerhead ribozyme sequences were incorporated into a tyrosine tRNA (tRNA(Tyr)) and compared with nonembedded molecules. To increase the levels of ribozyme and control antisense in vivo, sequences were expressed from an autonomously replicating vector derived from African cassava mosaic geminivirus. In vitro, the nonembedded ribozyme cleaved more target RNA, encoding chloramphenicol acetyltransferase (CAT), than the tRNA(Tyr) ribozyme. In contrast, the tRNA(Tyr) ribozyme was considerably more effective in vivo than either the nonembedded ribozyme or antisense sequences, reducing CAT activity to < 20% of the control level. A target sequence (CM2), mutated to be noncleavable, showed no reduction in CAT activity in the presence of the tRNA(Tyr) ribozyme beyond that for the antisense construct. The reduction in full-length CAT mRNA and the presence of specific cleavage products demonstrated in vivo cleavage of the target mRNA by the tRNA(Tyr) ribozyme. The high titer of tRNA(Tyr) ribozyme was a result of transcription from the RNA polymerase III promoter and led to the high ribozyme/substrate ratio essential for ribozyme efficiency.

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.