J. Christopher States

A summary of mutations in the UV-sensitive disorders: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.

The human diseases xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy are caused by mutations in a set of interacting gene products, which carry out the process of nucleotide excision repair. The majority of the genes have now been cloned and many mutations in the genes identified. The relationships between the distribution of mutations in the genes and the clinical presentations can be used for diagnosis and for understanding the functions and the modes of interaction among the gene products. The summary presented here represents currently known mutations that can be used as the basis for future studies of the structure, function, and biochemical properties of the proteins involved in this set of complex disorders, and may allow determination of the critical sites for mutations leading to different clinical manifestations. The summary indicates where more data are needed for some complementation groups that have few reported mutations, and for the groups for which the gene(s) are not yet cloned. These include the Xeroderma pigmentosum (XP) variant, the trichothiodystrophy group A (TTDA), and ultraviolet sensitive syndrome (UVs) groups. We also recommend that the XP‐group E should be defined explicitly through molecular terms, because assignment by complementation in culture has been difficult. XP‐E by this definition contains only those cell lines and patients that have mutations in the small subunit, DDB2, of a damage‐specific DNA binding protein. Hum Mutat 14:9–22, 1999.

Evidence for increased translational efficiency in the induction of P450IIE1 by solvents: Analysis of P450IIE1 mRNA polyribosomal distribution

The potential for enhanced translational processing of P450IIE1 mRNA during the early phase of P450IIE1 induction by pyridine or acetone was assessed by hybridization analysis of polyribosomal P450IIE1 mRNA distribution in rat hepatic tissue. Optical absorbance profiles of polyribosomal fractions exhibited an apparent shift at 5 h following pyridine administration relative to control. Slot and Northern blot analyses for P450IIE1 mRNA in the cytoplasmic extracts isolated from 5 h pyridine-treated rats demonstrated a shift in distribution of P450IIE1 message toward heavier polyribosomal fractions and Northern blot analysis suggested the presence of different populations of P450IIE1 mRNA. Slot blot analyses also demonstrated a shift in the polyribosomal distribution of P450IIE1 mRNA at 12 h following pyridine treatment; in contrast, hybridization analysis for P450IA1 revealed no shift in polyribosomal distribution of P450IA1 mRNA. Acute acetone administration to animals also resulted in a similar shift in polyribosomal distribution of P450IIE1 mRNA as compared to control. These data suggest that P450IIE1 mRNA shifts toward larger polyribosomes following acute exposure of animals to pyridine or acetone and provide evidence that induction of P450IIE1 at early times following acute pyridine or acetone administration involves enhanced translational efficiency through increased loading of ribosomes on P450IIE1 mRNA.

p53 Suppression of Arsenite-Induced Mitotic Catastrophe Is Mediated by p21CIP1/WAF1

Arsenic trioxide, an acute promyelocytic leukemia chemotherapeutic, may be an efficacious treatment for other cancers. Understanding the mechanism as well as genetic and molecular characteristics associated with sensitivity to arsenite-induced cell death is key to providing effective chemotherapeutic usage of arsenite. Arsenite sensitivity correlates with deficient p53 pathways in multiple cell lines. The role of p53 in preventing arsenite-induced mitotic arrest-associated apoptosis (MAAA), a form of mitotic catastrophe, was examined in TR9-7 cells, a model cell line with p53 exogenously regulated in a tetracycline-off expression system. Arsenite activated G1 and G2 cell cycle checkpoints independently of p53, but mitotic catastrophe occurred preferentially in p53(-) cells. Cyclin B/CDC2(CDK1) stabilization and caspase-3 activation persisted in arsenite-treated p53(-) cells consistent with MAAA/mitotic catastrophe. N-Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a pan-caspase inhibitor, completely abolished arsenite-induced MAAA/mitotic catastrophe and greatly increased the mitotic index. WEE1 and p21CIP1/WAF1 inhibit cyclin B/CDC2 by CDC2 tyrosine-15 phosphorylation and direct binding, respectively. CDC2-Y15-P was transiently elevated in arsenite-treated p53(+) cells but persisted in p53(-) cells. Arsenite induced p53-S15-P and p21CIP1/WAF1 only in p53(+) cells. P21CIP1/WAF1-siRNA-treated p53(+) cells were similar to p53(-) cells in mitotic index and cell cycle protein levels. p53-inducible proteins GADD45α and 14-3-3σ are capable of inhibiting cyclin B/CDC2 but did not play a p53-dependent role in mitotic escape in TR9-7 cells. The data indicate that p53 mediates cyclin B/CDC2 inactivation and mitotic release directly via p21CIP1/WAF1 induction.

Phosphorylation and Activation of Brain Tryptophan Hydroxylase: Identification of Serine‐58 as a Substrate Site for Protein Kinase A

Abstract: Tryptophan hydroxylase, the initial and rate‐limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is activated by protein kinase A and calcium/calmodulin‐dependent protein kinase. One important aspect of the regulation of any enzyme by a phosphorylation‐dephosphorylation cascade, and one that is lacking for tryptophan hydroxylase, lies in the identification of its site of phosphorylation by protein kinases. Recombinant forms of brain tryptophan hydroxylase were expressed as glutathione S‐transferase fusion proteins and exposed to protein kinase A. This protein kinase phosphorylates and activates full‐length tryptophan hydroxylase. The inactive regulatory domain of the enzyme (corresponding to amino acids 1–98) was also phosphorylated by protein kinase A. The catalytic core of the hydroxylase (amino acids 99–444), which expresses high levels of enzyme activity, was neither phosphorylated nor activated by protein kinase A. Conversion of serine‐58 to arginine resulted in the expression of a full‐length tryptophan hydroxylase mutant that, although remaining catalytically active, was neither phosphorylated nor activated by protein kinase A. These results indicate that the activation of tryptophan hydroxylase by protein kinase A is mediated by the phosphorylation of serine‐58 within the regulatory domain of the enzyme.

Enhanced XPA mRNA levels in cisplatin-resistant human ovarian cancer are not associated with XPA mutations or gene amplification

Enhanced expression of the nucleotide excision repair gene XPA is associated with resistance to cisplatin treatment in human ovarian cancer. Understanding the cause of enhanced XPA expression will provide new molecular targets for therapy directed at overcoming chemoresistance. Enhanced gene expression in cancer cells is often caused by mutations or gene amplification. Molecular analyses of the XPA genes in human ovarian cancers indicate that gene mutation and amplification are not the cause of enhanced XPA mRNA levels in ovarian cancers overexpressing XPA. Altered nucleotide excision repair (NER) gene regulation in chemoresistant tumors is discussed.

Tryptophan Hydroxylase: Cloning and Expression of the Rat Brain Enzyme in Mammalian Cells

Abstract: A cDNA encoding full‐length tryptophan hydroxylase was produced by reverse transcriptase‐PCR from rat brain mRNA and expressed transiently in a human fibroblast cell line. Catalytic activity was low unless transfected cells were grown in the presence of FeSO4. Recombinant tryptophan hydroxylase was found almost exclusively within the soluble compartment of the cell and was dependent on tryptophan and tetrahydrobiopterin for activity. The catalytic activity of recombinant tryptophan hydroxylase was stimulated >25‐fold by Fe(II) and to a somewhat lesser extent by the polyanions heparin and phosphatidylserine. The enzyme was inhibited by desferrioxamine and dopamine, both of which complex iron. When extracts from transfected cells were subjected to sucrose gradient centrifugation and analytical gel filtration, the recombinant enzyme behaved the same as the native enzyme from brain. A monoclonal antibody against phenylalanine hydroxylase that cross‐reacts with brain tryptophan hydroxylase was capable of immunoprecipitating the recombinant hydroxylase from solution. These data indicate that recombinant tryptophan hydroxylase expressed in mammalian cells is assembled into tetramers of ∼220,000 daltons. Its catalytic and physical properties appear to be very similar to those of the native enzyme from brain.

Exit from arsenite-induced mitotic arrest is p53 dependent.

Background Arsenic is both a human carcinogen and a chemotherapeutic agent, but the mechanism of neither arsenic-induced carcinogenesis nor tumor selective cytotoxicity is clear. Using a model cell line in which p53 expression is regulated exogenously in a tetracycline-off system (TR9-7 cells), our laboratory has shown that arsenite disrupts mitosis and that p53-deficient cells [p53(−)], in contrast to p53-expressing cells [p53(+)], display greater sensitivity to arsenite-induced mitotic arrest and apoptosis. Objective Our goal was to examine the role p53 plays in protecting cells from arsenite-induced mitotic arrest. Methods p53(+) and p53(−) cells were synchronized in G2 phase using Hoechst 33342 and released from synchrony in the presence or absence of 5 μM sodium arsenite. Results Mitotic index analysis demonstrated that arsenite treatment delayed exit from G2 in p53(+) and p53(−) cells. Arsenite-treated p53(+) cells exited mitosis normally, whereas p53(−) cells exited mitosis with delayed kinetics. Microarray analysis performed on mRNAs of cells exposed to arsenite for 0 and 3 hr after release from G2 phase synchrony showed that arsenite induced inhibitor of DNA binding-1 (ID1) differentially in p53(+)and p53(−) cells. Immunoblotting con-firmed that ID1 induction was more extensive and sustained in p53(+) cells. Conclusions p53 promotes mitotic exit and leads to more extensive ID1 induction by arsenite. ID1 is a dominant negative inhibitor of transcription that represses cell cycle regulatory genes and is elevated in many tumors. ID1 may play a role in the survival of arsenite-treated p53(+) cells and contribute to arsenic carcinogenicity.

Splice site mutations in a xeroderma pigmentosum group A patient with delayed onset of neurological disease

XP12BE is a commonly studied XP-A cell line that exhibits slightly increased resistance to UV compared with the majority of XP-A cell lines. The elevated UV survival is common to a subset of XP-A cell lines and correlates with delayed onset of the neurological disease in patients. We identified the XPA mutations in XP12BE by single strand conformation polymorphism (SSCP) analyses and nucleotide sequencing. XP12BE is a compound heterozygote and both mutations affect mRNA splicing. One mutation is a G to C transversion within the splice donor site of intron 4 that is common to several cell lines from XP-A patients with delayed onset of neurological disease. The other mutation is a G to T transversion at the same position as a G to C transversion in the splice acceptor site of intron 3 that is common in Japanese XP-A patients. We also demonstrated the persistence of the XP12BE mutations in cell line 2-O-A2 which has been shown to express XPA protein. These results suggest that the intron 4 splice donor mutation likely produces some, at least partially functional, XPA protein that accounts for the increased UV survival of XP-A cell lines derived from patients with delayed onset of neurological disease.

Preferential DNA damage in the p53 gene by benzo[a]pyrene metabolites in cytochrome P4501A1-expressing xeroderma pigmentosum group A cells

Gene‐specific DNA damage levels were determined by quantitative polymerase chain reaction (QPCR) after treating cytochrome P450 (CYP) 1A1‐expressing xeroderma pigmentosum fibroblasts with [3H]benzo[a]pyrene‐trans‐7,8‐dihydrodiol ([3H]BPD) or [3H]benzo[a]pyrene‐trans‐7,8‐dihydrodiol‐9, 10‐epoxide([3H]BPDE). DNA damage in the p53 gene (which is transcriptionally active) and the β‐globin gene (which is transcriptionally inactive) was measured in cells treated with [3H](±)‐anti‐BPDE, [3H](±)‐BPD, and [3H](−)‐BPD. DNA adduct formation in the genome overall was determined by measuring the incorporation of 3H into DNA. DNA damage in a p53 gene fragment (exons 8–9, 445 bp) was readily detected by QPCR. DNA damage was either not detected or much reduced in a similarly sized target in the β‐globin gene (exons 1–2, 551 bp). At equivalent levels of genomic DNA adducts, BPD treatment induced more damage in the p53 gene than BPDE treatment did. The lesion frequencies in the p53 and β‐globin genes in purified DNA treated with BPDE in vitro were the same, indicating that there was no sequence‐specific basis for preferential lesion formation in the p53 gene in treated cells. DNA damage in both the p53 and β‐globin genes showed a dose response to [3H](−)‐BPD. The frequency of BPD‐induced lesions in the p53 gene was sixfold to sevenfold greater than in the β‐globin gene and 200‐ to 300‐fold greater than in bulk DNA. The BPD‐induced lesion frequency in the β‐globin gene was 30‐ to 50‐fold greater than in bulk DNA. The data indicate that the distribution of BPDE‐induced DNA lesions is dramatically nonrandom and suggest that the nonrandomness is governed by DNA sequence composition, chromatin structure, and dose rate.

Characterization of the human XPA promoter

We cloned the human xeroderma pigmentosum group A gene (XPA) and characterized the XPA promoter (pXPA) by transient cat expression. The pXPA is extraordinarily weak in human fibroblasts (1% of RSV-LTR) and appears to function without any of the usual promoter elements. Regions containing positive and negative control elements were localized.