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1.
Oncogene ; 27(28): 3977-85, 2008 Jun 26.
Article in English | MEDLINE | ID: mdl-18317453

ABSTRACT

The cellular response to the introduction of double strand DNA breaks involves complexes of protein interactions that govern cell cycle checkpoint arrest and repair of the DNA lesions. The checkpoint kinases Chk1 and Chk2 phosphorylate the carboxy-terminal domain of hBRCA2, a protein involved in recombination-mediated DNA repair (HRR) and replication fork maintenance. Cells deficient in hBRCA2 are hypersensitive to DNA damaging agents. Phosphorylation of the residue in hBRCA2 targeted by the Chk1 and Chk2 kinases regulates its interaction with Rad51. Furthermore, the cell line lex1/lex2, which lacks the carboxy-terminal domain containing the phosphorylated residue, does not support localization of Rad51 to nuclear foci after exposure to UV or treatment with ionizing radiation (IR). The data show that either phosphorylation of Rad51 by Chk1 or phosphorylation of the carboxy-terminal domain of hBRCA2 by Chk1 or Chk2 plays a critical role in the binding of Rad51 to hBRCA2 and the subsequent recruitment of Rad51 to sites of DNA damage. While depletion of Chk1 from cells leads to loss of Rad51 localization to nuclear foci in response to replication arrest, cells lacking Chk2 also show a defect in Rad51 localization, but only in presence of double strand DNA breaks, indicating that each of these kinases may contribute somewhat differently to the formation of Rad51 nucleoprotein filaments depending on the type of DNA damage incurred by the cells.


Subject(s)
BRCA2 Protein/physiology , DNA Damage , Gene Expression Regulation , Protein Kinases/physiology , Protein Serine-Threonine Kinases/physiology , Rad51 Recombinase/physiology , Apoptosis Regulatory Proteins , Checkpoint Kinase 1 , Checkpoint Kinase 2 , Humans , Microscopy, Confocal , Models, Biological , Models, Genetic , Nuclear Proteins/chemistry , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Ultraviolet Rays
2.
Mutat Res ; 614(1-2): 48-55, 2007 Jan 03.
Article in English | MEDLINE | ID: mdl-16914171

ABSTRACT

Mutation frequencies at some loci in mammalian somatic cells in vivo approach 10(-4). The majority of these events occur as a consequence of loss of heterozygosity (LOH) due to mitotic recombination. Such high levels of DNA damage in somatic cells, which can accumulate with age, will cause injury and, after a latency period, may lead to somatic disease and ultimately death. This high level of DNA damage is untenable for germ cells, and by extrapolation for embryonic stem (ES) cells, that must recreate the organism. ES cells cannot tolerate such a high frequency of damage since mutations will immediately impact the altered cell, and subsequently the entire organism. Most importantly, the mutations may be passed on to future generations. ES cells, therefore, must have robust mechanisms to protect the integrity of their genomes. We have examined two such mechanisms. Firstly, we have shown that mutation frequencies and frequencies of mitotic recombination in ES cells are about 100-fold lower than in adult somatic cells or in isogenic mouse embryonic fibroblasts (MEFs). A second complementary protective mechanism eliminates those ES cells that have acquired a mutational burden, thereby maintaining a pristine population. Consistent with this hypothesis, ES cells lack a G1 checkpoint, and the two known signaling pathways that mediate the checkpoint are compromised. The checkpoint kinase, Chk2, which participates in both pathways is sequestered at centrosomes in ES cells and does not phosphorylate its substrates (i.e. p53 and Cdc25A) that must be modified to produce a G1 arrest. Ectopic expression of Chk2 does not rescue the p53-mediated pathway, but does restore the pathway mediated by Cdc25A. Wild type ES cells exposed to ionizing radiation do not accumulate in G1 but do so in S-phase and in G2. ES cells that ectopically express Chk2 undergo cell cycle arrest in G1 as well as G2, and appear to be protected from apoptosis.


Subject(s)
Embryonic Stem Cells/metabolism , Mutation , Adenine Phosphoribosyltransferase/deficiency , Adenine Phosphoribosyltransferase/genetics , Animals , Checkpoint Kinase 2 , DNA Damage , DNA Repair , Embryonic Stem Cells/radiation effects , Genome , Mice , Mice, Knockout , Models, Genetic , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Recombination, Genetic , Signal Transduction , Transfection
3.
Kidney Int ; 60(3): 910-23, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11532086

ABSTRACT

BACKGROUND: We have previously shown that, as in human adenine phosphoribosyltransferase (APRT) deficiency, Aprt knockout mice form 2,8-dihydroxyadenine (DHA) renal stones. The disease develops earlier and is more severe in male than in female mice. To examine the biological bases for these differences, the area occupied by DHA crystals was quantified in kidney sections from male and female mice (strain 129) aged one day to eight months and this parameter was correlated with changes in renal histopathology. Aprt heterozygous and wild-type mice were used as controls. METHODS: Following anesthesia, the left kidney was removed and immediately frozen in dry ice. Unstained cryosections were examined by polarized light to determine total area of birefringent particles. The right kidney was perfused and embedded in plastic, and stained sections were viewed by light microscopy to examine the histopathology and to determine the location of the birefringent particles. A pathological score was assigned to the histological findings. The scores from the right kidney were compared with crystal/particle area in the left kidney, and the data were analyzed using two-way analysis of variance. The chemical composition of the particles was determined by x-ray diffraction analysis. Several stone fragments from the bladder were also examined by scanning electron microscopy (SEM). RESULTS: Crystals were detected in kidney sections from one- to two-day-old Aprt knockout mice. The crystal burden remained low in both sexes throughout the study except in males at the 120- to 240-day period. Furthermore, there was a substantial degree of renal pathology, primarily seen as interstitial fibrosis, in those males with a very high level of stone formation. The crystalline material was identified as 6-amino-2,8(3,9)-purine dione, a tautomeric form of DHA. SEM indicated that the crystals were spherical, with a diameter of 10 to 20 microm. Tissue staining and fixation procedures dramatically reduced the amount of birefringent material in kidney sections. Aprt heterozygotes of both sexes had low levels of crystalline material in the kidneys and no pathology. Birefringent material or pathological changes were not seen in kidneys from wild-type mice. CONCLUSIONS: Both male and female Aprt knockout mice accumulate DHA. However, the area occupied by DHA crystals was significantly greater in 120- to 240-day-old males compared with the females of similar age. Also, substantial renal pathology was detected in kidneys of male mice that had very high levels of stone material.


Subject(s)
Adenine Phosphoribosyltransferase/deficiency , Adenine/analogs & derivatives , Kidney Calculi/genetics , Kidney Tubules, Distal/pathology , Kidney Tubules, Proximal/pathology , Adenine/metabolism , Adenine Phosphoribosyltransferase/genetics , Age Factors , Animals , Female , Genotype , Kidney Calculi/metabolism , Kidney Calculi/pathology , Kidney Tubules, Distal/chemistry , Kidney Tubules, Proximal/chemistry , Male , Mice , Mice, Knockout , Microscopy, Electron, Scanning , Sex Factors , Tissue Fixation , X-Ray Diffraction
4.
Mol Biol Cell ; 12(8): 2352-63, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11514621

ABSTRACT

The AP-1 transcription factor is activated by oncogenic signal transduction cascades and its function is critical for both mitogenesis and carcinogenesis. To define the role of AP-1 in the context of a human fibrosarcoma cell line, HT1080, we expressed a dominant negative c-jun mutant fused to the green fluorescent protein in an ecdysone-inducible system. We demonstrated that high levels of this mutant, GFP-TAM67, inhibit AP-1 activity and arrest cells predominantly in the G1 phase of the cell cycle. This arrest is reversible and occurs only above a threshold concentration; low to moderate levels of GFP-TAM67 are insufficient for growth arrest. Contrary to expectations based on the literature, GFP-TAM67 does not inhibit expression of cyclin D1, cyclin E, or their respective cyclin-dependent kinases. However, pRB is hypophosphorylated in GFP-TAM67-arrested cells and the activity of both the cyclin D1:cdk and the cyclin E:cdk complexes are impaired. Both of these complexes show an increased association with p21(CIP1/WAF1), concomitantly with induction of the p21 mRNA by GFP-TAM67. These results suggest a novel function of AP-1 in the activation of the G1 cyclin:cdk complexes in human tumor cells by regulating the expression of the p21(CIP1/WAF1) gene.


Subject(s)
Cyclin D1/metabolism , Cyclin E/metabolism , Cyclin-Dependent Kinases/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Transcription Factor AP-1/metabolism , Cell Cycle/physiology , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , Cyclins/metabolism , Ecdysone/analogs & derivatives , Ecdysone/genetics , Enzyme Inhibitors/metabolism , Flow Cytometry , Genes, Reporter , Humans , Immunoblotting , Microscopy, Confocal , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-jun/genetics , Recombinant Fusion Proteins/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Transcription Factor AP-1/genetics , Tumor Cells, Cultured
5.
Nat Genet ; 28(2): 169-72, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11381266

ABSTRACT

Mitotic recombination occurs with high frequency in humans and mice. It leads to loss of heterozygosity (LOH) at important gene loci and can cause disease. However, the genetic modulators of mitotic recombination are not well understood. As recombination depends on a high level of nucleotide sequence homology, we postulate that the frequency of somatic variants derived from mitotic recombination should be diminished in progeny from crosses between strains of mice in which nucleotide sequences have diverged. Here we report that mitotic recombination is suppressed, to various degrees in different tissues, in hybrids of distantly related mouse strains. Reintroduction of greater chromosomal homology by backcrossing restores mitotic recombination in offspring. Thus, chromosomal divergence inhibits mitotic recombination and, consequently, may act as a modifier of cancer susceptibility by limiting the rate of LOH. The suppression of mitotic recombination in some F1 hybrids in which meiotic recombination persists indicates that these processes are differentially affected by chromosomal divergence.


Subject(s)
Adenine Phosphoribosyltransferase/genetics , Mice, Inbred Strains/genetics , Mitosis , Recombination, Genetic , Animals , Chimera , Chromosomes , Crosses, Genetic , Female , Loss of Heterozygosity , Male , Mice , Mice, Inbred C57BL
6.
Laryngoscope ; 110(11): 1798-801, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11081587

ABSTRACT

OBJECTIVES/HYPOTHESIS: There is currently no single histological or genotypic marker that reliably predicts the biological behavior of head and neck squamous cell carcinoma (HNSCC). While multiple genetic mutations have been investigated, no single genotypic alteration has consistently correlated with tumor aggressiveness. Phenotypic markers may prove more predictive, because they can represent many different genetic alterations. We investigated the frequency of centrosome hyperamplification in HNSCC and examined its usefulness as a marker for tumor recurrence. STUDY DESIGN: Analysis of archived paraffin blocks using immunohistochemistry. METHODS: Eighteen patients who underwent resection of oral cavity squamous cell carcinoma were reviewed. Ten patients had cancers that recurred locally within 1 year of resection, and 8 patients were tumor free at 5 years. The amount of centrosome hyperamplification in the cancer specimens and all surgical margins was graded as follows: 0, none; 1+, rare hyperamplification; 2+, greater than 10% of cells per high-powered field; and 3 +, greater than 20% of cells per high-powered field. RESULTS: Centrosome hyperamplification was found in 17 of 18 tumors (94%). Grade 2+ or 3+ hyperamplification was found more in cancers that recurred (9 of 10) than in those that did not (3 of 8) and was more prevalent in the histologically normal margins of patients with recurrence (8 of 10) than in those without recurrent cancer (3 of 8). CONCLUSIONS: Our results demonstrate the extremely frequent occurrence of centrosome hyperamplification in HNSCC. Centrosome hyperamplification is a phenotypic marker for HNSCC and can reflect multiple genotypic changes. Its presence in histologically normal margins suggests that it may be useful for analysis of primary tumors and tumor margins.


Subject(s)
Carcinoma, Squamous Cell/ultrastructure , Centrosome/pathology , Mouth Neoplasms/ultrastructure , Adult , Aged , Aged, 80 and over , Carcinoma, Squamous Cell/mortality , Female , Humans , Immunohistochemistry , Male , Middle Aged , Mouth Neoplasms/mortality , Neoplasm Staging , Phenotype , Prognosis
7.
Kidney Int ; 58(2): 528-36, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10916076

ABSTRACT

BACKGROUND: We have developed a knockout mouse model for adenine phosphoribosyltransferase (APRT) deficiency, a condition that often leads to 2,8-dihydroxyadenine (DHA) nephrolithiasis in humans. Aprt knockout male mice develop severe renal damage by three months of age, but this is strain specific. Renal damage in female mice is less pronounced than in males. The gene level changes that promote renal injury in APRT-deficient mice are not known. METHODS: We used mRNA differential display polymerase chain reaction (DD-PCR) to analyze renal gene expression changes in APRT-deficient male and female mice (strain C3H) compared with age- and sex-matched Aprt heterozygote controls. The differentially amplified bands were reamplified, cloned, sequenced, and queried against the National Center for Biotechnology Information nonredundant databases using the Basic Alignment Search Tool. Relative quantitative reverse transcription-polymerase chain reaction was used to confirm the results of DD-PCR for a selected number of genes in one-, three-, and six-month-old male and female mice. RESULTS: Sixty-three differentially amplified bands were identified, including 21 for known genes, and 8 of these were examined further. In three-month-old APRT-deficient male mice, the expression of C10 was increased tenfold, and there was a fourfold to sevenfold increase in the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-1), MGP (matrix Gla protein), and lysyl oxidase (LOX). The expression of cholecystokinin-A receptor (CCKAR), imprinted multimembrane-spanning polyspecific transporter-like gene 1 (IMPT-1), and kidney androgen-regulated protein (KAP) was diminished twofold to fourfold, but there was little or no change in the expression of organic anion transporter (OATP). Except for a more than tenfold increase in C10 expression and up to tenfold decrease in KAP expression, APRT-deficient female mice did not show significant changes in gene expression compared with controls. CONCLUSIONS: These findings suggest that (1) there are sex-related differences in gene expression in DHA lithiasis, possibly caused by increased deposition of DHA crystals in male compared with female kidneys; and (2) the expression of certain genes (for example, C10) may simply be an indication of nonspecific cellular stimulation and may not be related to renal injury.


Subject(s)
Adenine Phosphoribosyltransferase/genetics , Adenine/analogs & derivatives , Kidney Calculi/physiopathology , Kidney/physiology , Adenine/metabolism , Adenine Phosphoribosyltransferase/deficiency , Age Factors , Animals , DNA Primers , DNA, Complementary , Disease Models, Animal , Female , Gene Expression/physiology , Kidney Calculi/enzymology , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/analysis , Sex Characteristics
8.
Oncogene ; 19(34): 3948-54, 2000 Aug 10.
Article in English | MEDLINE | ID: mdl-10951588

ABSTRACT

Activating mutations of RAS are thought to be early events in the evolution of thyroid follicular neoplasms. We used a doxycycline-inducible expression system to explore the acute effects of H-RAS12 on genomic stability in thyroid PCCL3 cells. At 2-3 days (first or second cell cycle) there was a significant increase in the frequency of micronucleation. Treatment of cells with YVAD-CHO inhibited RAS-induced apoptosis, but had no effect on micronucleation. The effects of H-RAS(V12) were mediated by activation of MAPK, as treatment with PD98059 at concentrations verified to selectively inhibit MEK1 reduced the frequency of prevalence of cells with micronuclei. In addition, doxycycline-inducible expression of a constitutively active MEK1, but not of a mutant RAC1, mimicked the effects of H-RAS(V12). The effects of H-RAS(V12) on genome destabilization were apparent even though the sequence of p53 in PCCL3 cells was confirmed to be wild-type. Acute activation of H-RAS(V12) evoked a proportional increase in both CREST negative and CREST positive micronuclei, indicating that both clastogenic and aneugenic effects were involved. H-RAS(V12) and activated MEK1 also induced centrosome amplification, and chromosome misalignment. Evidence that acute expression of constitutively activated RAS destabilizes the genome of PCCL3 cells is consistent with a mode of tumor initiation in which this oncogene promotes phenotypic progression by predisposing to large scale genomic abnormalities.


Subject(s)
MAP Kinase Signaling System , Saccharomyces cerevisiae Proteins , Thyroid Neoplasms/genetics , ras Proteins/genetics , Animals , Apoptosis/radiation effects , Cell Nucleus/drug effects , Cell Nucleus/genetics , Chromosomal Proteins, Non-Histone/metabolism , Doxycycline/pharmacology , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Gamma Rays , Gene Expression Regulation, Neoplastic , MAP Kinase Kinase 1 , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/genetics , Mutation , Phosphoprotein Phosphatases/genetics , Protein Phosphatase 2 , Protein Phosphatase 2C , Protein Serine-Threonine Kinases/genetics , Rats , Thyroid Neoplasms/drug therapy , Thyroid Neoplasms/metabolism , Tumor Cells, Cultured , rac1 GTP-Binding Protein/genetics
9.
Cancer Res ; 60(13): 3404-8, 2000 Jul 01.
Article in English | MEDLINE | ID: mdl-10910047

ABSTRACT

Heritable gene silencing is an important mechanism of tumor suppressor gene inactivation in a variety of human cancers. In the present study, we show that methylation-associated silencing of the autosomal adenine phosphoribosyltransferase (Aprt) locus occurs in primary mouse kidney cells. Aprt-deficient cells were isolated from mice that were heterozygous for Aprt, i.e., they contained one wild-type Aprt allele and one targeted allele bearing an insertion of the bacterial neo gene. Although silencing of the wild-type allele alone was sufficient for the cells to become completely Aprt-deficient, biallelic methylation of the promoter region was found to occur. Moreover, despite the absence of selective pressure against the targeted allele, phenotypic silencing of the inserted neo gene accompanied silencing of the wild-type Aprt allele. A potential role for allelic homology in these events is discussed.


Subject(s)
Adenine Phosphoribosyltransferase/genetics , DNA Methylation , Gene Silencing , Kidney/enzymology , Promoter Regions, Genetic , Adenine/pharmacology , Animals , Azaserine/pharmacology , Clone Cells , Gene Expression Regulation, Enzymologic/drug effects , Gene Silencing/drug effects , Heterozygote , Humans , Hypoxanthine Phosphoribosyltransferase/deficiency , Hypoxanthine Phosphoribosyltransferase/genetics , Kanamycin Kinase/genetics , Mice , Mice, Transgenic , Restriction Mapping
10.
Proc Natl Acad Sci U S A ; 97(13): 7405-10, 2000 Jun 20.
Article in English | MEDLINE | ID: mdl-10861008

ABSTRACT

The p53 tumor suppressor protein participates in multiple cellular processes including cell cycle checkpoints and programmed cell death. In cell lines, loss of p53 function is associated with increased genetic instability including aneuploidy, gene amplification, and point mutation. Although similar genetic instability often accompanies the progression of malignancy in tumors, its role in tumor initiation in normal cells is not clear. To study whether or not loss of p53 leads to genetic instability in normal cells in vivo, we have examined mechanisms of loss of heterozygosity (LOH) at the Aprt (adenine phosphoribsyltransferase) and flanking loci in normal fibroblasts and T lymphocytes of p53-deficient mice. Somatic cell variants that arose in vivo as a consequence of genetic or epigenetic alterations abolishing Aprt function were selected and expanded in vitro by virtue of their resistance to 2,6-diaminopurine (DAP). We observed that p53 null mice produced about three times as many DAP-resistant fibroblast colonies than wild-type mice, but the frequency of DAP-resistant T lymphocyte colonies was not significantly changed. Mitotic recombination, but not point mutation, partly accounted for the increase in the frequency of DAP-resistant fibroblasts. Most significantly, chromosome loss/duplication and interstitial deletion, which were extremely rare events in the wild-type mice, represented a significant proportion of LOH events in both fibroblasts and T lymphocytes of p53 null mice. Also, increased interstitial deletion was observed in fibroblasts of p53 heterozygous mice. These data suggest that increased genetic variation, including chromosome instability, starts at the initiation stage of tumorigenesis when functional p53 is absent or reduced.


Subject(s)
Genes, p53 , Loss of Heterozygosity , Tumor Suppressor Protein p53/genetics , Aneuploidy , Animals , Gene Amplification , Mice , Mice, Knockout , Point Mutation
11.
Cancer Gene Ther ; 7(2): 240-6, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10770632

ABSTRACT

Delivery and expression of the herpes simplex virus thymidine kinase (HSVtk) gene in combination with the prodrug ganciclovir is currently being evaluated for the treatment of many types of cancer. After initial phosphorylation by HSVtk, cellular kinases generate the toxic triphosphate form of ganciclovir (GCV). To further define the role of GCV metabolism in cells expressing HSVtk, two human tumor cell lines, UMSCC29 and T98G, were transduced with HSVtk and screened for insertion of one or two copies of the viral transgene by Southern blot analysis. Both the relative capacities for incorporating labeled GCV and the levels of GCV metabolites were determined for each of the parental cell lines and their derivatives containing either one or two copies of the HSVtk gene. The efficiency of GCV killing and the magnitude of the bystander effect were compared for the single- and double-copy HSVtk cell lines. Consistently, cells that expressed two copies of HSVtk metabolized GCV more efficiently, were more sensitive to GCV, and demonstrated improved bystander killing relative to single-copy HSVtk cells. The implications of these results for future and current therapies employing HSVtk and GCV are discussed.


Subject(s)
Antiviral Agents/toxicity , Ganciclovir/toxicity , Simplexvirus/enzymology , Simplexvirus/genetics , Thymidine Kinase/genetics , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/enzymology , Antiviral Agents/metabolism , Cell Death/drug effects , Cell Death/genetics , Ganciclovir/metabolism , Gene Dosage , Humans , Phosphorylation , Simplexvirus/drug effects , Tumor Cells, Cultured/pathology
12.
Environ Mol Mutagen ; 35(2): 150-7, 2000.
Article in English | MEDLINE | ID: mdl-10712749

ABSTRACT

We have used B6C3F1 mice heterozygous at Aprt (adenine phosphoribosyltransferase) as a model to study in vivo loss of heterozygosity (LOH) in normal splenic T-lymphocytes. APRT-deficient T-cells were selected in medium containing 50 microg/ml 2, 6-diaminopurine (DAP), an adenine analog that is toxic only to cells with APRT enzyme activity. DAP-resistant (DAP(r)) T-cell variants were recovered at an average frequency of 3 x 10(-5) from 21 B6C3F1 Aprt(+/-) mice. Allele-specific PCR of Aprt showed that about 70% of 122 DAP(r) colonies were caused by loss of the nontargeted Aprt allele (Aprt(+)). Analysis of microsatellite markers along the length of chromosome 8 suggested that mitotic recombination, or chromosome loss, with or without duplication of the remaining chromosome are the predominant mechanisms resulting in loss of Aprt(+). DNA sequencing of Aprt RT-PCR products from the DAP(r) variants that retained Aprt(+) indicated that point mutation as well as other mechanisms could cause this second class of variants. The high spontaneous frequency of in vivo Aprt LOH in mouse T-cells, mediated by LOH mechanisms that are also known to produce human cancers, suggests that the Aprt heterozygous mouse is a valid model for studying the diversity of mechanisms for in vivo somatic mutagenesis.


Subject(s)
Adenine Phosphoribosyltransferase/genetics , Loss of Heterozygosity , T-Lymphocytes/enzymology , Animals , Base Sequence , DNA Primers , Humans , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Polymerase Chain Reaction
13.
Cancer Res ; 60(24): 6826-31, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11156373

ABSTRACT

The polo-like kinases (Plks) are a family of conserved serine/threonine kinases that play a critical role in the normal progression of cells through mitosis. The Plk3 serine/threonine kinase is a mammalian member of this family. Overexpression of Plk3 in mammalian cells suppresses proliferation and inhibits colony formation. Subsequent analysis demonstrated that overexpression of Plk3 induces chromatin condensation and apoptosis. This phenotype could not be inhibited by coexpression of Bcl-2 and was partially dependent on the COOH-terminal domain of Plk3 but not on the catalytic activity of Plk3. Analysis of EGFP-Plk3 subcellular localization revealed that Plk3 localizes to the cellular cortex and to the cell midbody during exit from mitosis and is consistent with a role in cytokinesis. These data suggest that overexpression or ectopic suppression of Plk3 interferes with cellular proliferation by impeding cytokinesis.


Subject(s)
Apoptosis , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/physiology , Cell Division , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/physiology , Alleles , Annexin A5/metabolism , Blotting, Western , Cell Line , Chromatin/metabolism , Dose-Response Relationship, Drug , Green Fluorescent Proteins , HeLa Cells , Humans , Luminescent Proteins/metabolism , Microscopy, Confocal , Microscopy, Fluorescence , Mitosis , Mutagenesis, Site-Directed , Phenotype , Precipitin Tests , Protein Synthesis Inhibitors/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Puromycin/pharmacology , Temperature , Time Factors , Transfection , Tumor Suppressor Proteins
14.
J Biol Chem ; 274(53): 38083-90, 1999 Dec 31.
Article in English | MEDLINE | ID: mdl-10608877

ABSTRACT

The generation of micronuclei is a reflection of DNA damage, defective mitosis, and loss of genetic material. The involvement of the MAPK pathway in mediating v-ras-induced micronuclei in NIH 3T3 cells was examined by inhibiting MAPK activation. Conversely, the MAPK pathway was constitutively activated by infecting cells with a v-mos retrovirus. Micronucleus formation was inhibited by the MAPK kinase inhibitors PD98059 and U0126, but not by wortmannin, an inhibitor of the Ras/phosphatidylinositol 3-kinase pathway. Transduction of cells with v-mos resulted in an increase in micronucleus formation, also consistent with the involvement of the MAPK pathway. Staining with the anti-centromeric CREST antibody revealed that instability induced by constitutive activation of MAPK is due predominantly to aberrant mitotic segregation, since most of the micronuclei were CREST-positive, reflective of lost chromosomes. A significant fraction of the micronuclei were CREST-negative, reflective of lost acentric chromosome fragments. Some of the instability observed was due to mitotic events, consistent with the increased formation of bi-nucleated cells, which result from perturbations of the mitotic spindle and failure to undergo cytokinesis. This chromosome instability, therefore, is a consequence of mitotic aberrations, mediated by the MAPK pathway, including centrosome amplification and formation of mitotic chromosome bridges.


Subject(s)
Chromosome Deletion , Mitogen-Activated Protein Kinases/metabolism , Oncogene Protein p21(ras)/physiology , 3T3 Cells , Animals , Cell Transformation, Viral , Enzyme Activation , Enzyme Inhibitors/pharmacology , Leukemia Virus, Murine/physiology , MAP Kinase Signaling System , Mice , Micronuclei, Chromosome-Defective , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitosis/genetics , Oncogene Proteins v-mos/genetics , Phosphorylation
15.
Mutat Res ; 429(2): 225-37, 1999 Oct 19.
Article in English | MEDLINE | ID: mdl-10526207

ABSTRACT

Mammalian genomes are in constant jeopardy of invasion by prokaryotic DNA sequences because of their extensive exposure to bacteria; however, mammalian genomes appear to be protected from horizontal transmission of bacterial DNA. Transgenic mice provide a convenient model system for investigating the capacity of mammalian genomes in vivo to retain, silence, and/or reject foreign DNAs. We have previously reported that bacterial genes encoding the Lac repressor (lacI) are subject to sequence-dependent methylation and silencing in the transgenic mouse. In this paper, we report that bacterially derived lacI transgenes, but not their mammalian counterparts, can also be eliminated from the somatic cell DNA of affected animals. This somatic instability is heritable, strain-dependent, and conferred in cis. Our data are consistent with a model of genome surveillance in the mouse which can lead to loss of foreign DNA and which may be analogous to restriction-modification systems that maintain the integrity of the bacterial genome.


Subject(s)
DNA, Bacterial/genetics , Escherichia coli Proteins , Gene Deletion , Genome , Animals , Bacterial Proteins/genetics , Blotting, Southern , Crosses, Genetic , DNA, Bacterial/analysis , Female , Lac Repressors , Male , Mice , Mice, Transgenic , Models, Genetic , Pedigree , Repressor Proteins/genetics , Transgenes/genetics
16.
Cancer Res ; 59(19): 4781-3, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10519383

ABSTRACT

The presence of increased frequencies of blood-derived and solid tumors in ataxia-telangiectasia (A-T) patients, coupled with a role for the ATM (A-T mutation) protein in detecting specific forms of DNA damage, has led to the assumption of a mutator phenotype in A TM-deficient cells. Supporting this assumption are observations of increased rates of chromosomal aberrations and intrachromosomal homologous recombinational events in the cells of A-T patients. We have bred mice with knockout mutations for the selectable Aprt (adenine phosphoribosyltransferase) locus and the Atm locus to examine the frequency of second-step autosomal mutations in Atm-deficient cells. Two solid tissues were examined: (a) the ear, which yields predominately mesenchymal cells; and (b) the kidney, which yields predominately epithelial cells. We report here the lack of a mutator phenotype for inactivating autosomal mutations in solid tissues of the Atm-deficient mice.


Subject(s)
Adenine Phosphoribosyltransferase/genetics , Mutation , Protein Serine-Threonine Kinases , Proteins/genetics , Animals , Ataxia Telangiectasia/genetics , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins , Chromosome Mapping , DNA-Binding Proteins , Ear , Female , Genotype , Heterozygote , Homozygote , Kidney/enzymology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Organ Specificity , Proteins/metabolism , Tumor Suppressor Proteins
17.
Arch Otolaryngol Head Neck Surg ; 125(9): 949-52, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10488977

ABSTRACT

OBJECTIVE: To evaluate multiple genetic loci in patients with head and neck cancer to determine if, as in colorectal carcinoma, there is an orderly occurrence of genetic alterations, and if an accumulation of alterations affects patient survival. DESIGN: Cohort study of patients with head and neck cancer in which fresh tissue was retrieved. SETTING: Academic medical center. PATIENTS: Forty-three patients treated surgically for squamous cell carcinoma of the head and neck from 1991 to 1994. MAIN OUTCOME MEASURES: The DNA from tumor and healthy tissue was evaluated for loss of heterozygosity at p53, retinoblastoma, and chromosome 16q and for amplification of cyclin D1. The respective RNA was probed for levels of p53, p 16, p21, and p27 messenger RNA. These findings were compared with tumor stage and patient survival. RESULTS: DNA analysis showed that loss of heterozygosity occurred at p53 in 21% of tumors, at retinoblastoma in 35%, and at 16q in 21%, and that cyclin D1 was amplified in 42%. Messenger RNA levels of the assessed proteins were variably increased and decreased compared with healthy tissues obtained from the same patients with no discernable pattern. There was no correlation between any one of these genetic alterations and overall survival. When patients were analyzed for loss of heterozygosity at p53, retinoblastoma, 16q, or altered cyclin D1 in combination, 19 patients had no detectable alterations, 13 had 1, 6 had 2, and 5 had 3. Single genetic alterations did not affect survival; however, there was a trend toward decreased survival with multiple alterations. The 2-year Kaplan-Meier survival in patients with less than 1 genetic loss was 78% vs 58% in patients with 2 or more losses. CONCLUSIONS: The lack of a pattern of genetic alterations in head and neck cancer demonstrates that its progression can be mediated by a multitude of pathways, complicating its genetic evaluation. Single genetic alterations do not appear to affect survival; however, when multiple alterations are detected-regardless of combination-survival is affected. This observation lends credence to the theory that multiple genetic alterations contribute to cancer progression; however, the lack of a pattern of this genetic change is a significant obstacle to applying genetic findings to routine cancer therapy.


Subject(s)
Carcinoma, Squamous Cell/genetics , Chromosome Aberrations/genetics , Chromosome Mapping , Otorhinolaryngologic Neoplasms/genetics , Adult , Aged , Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/mortality , Carcinoma, Squamous Cell/surgery , Cohort Studies , Colorectal Neoplasms/genetics , Colorectal Neoplasms/mortality , Colorectal Neoplasms/surgery , Female , Heterozygote , Humans , Male , Middle Aged , Otorhinolaryngologic Neoplasms/mortality , Otorhinolaryngologic Neoplasms/surgery , Prognosis , RNA, Messenger/genetics , Reference Values , Survival Rate
18.
Proc Natl Acad Sci U S A ; 96(16): 9230-5, 1999 Aug 03.
Article in English | MEDLINE | ID: mdl-10430925

ABSTRACT

Mice heterozygous at Aprt (adenine phosphoribosyltransferase) were used as a model to study in vivo loss of heterozygosity (LOH) in normal fibroblasts. Somatic cell variants that exhibited functional loss of the wild-type Aprt in vivo were recovered as APRT-deficient cell colonies after culturing in selection medium containing 2, 6-diaminopurine (DAP), an adenine analog that is toxic only to cells with APRT enzyme activity. DAP-resistant (DAP(r)) fibroblast variants were recovered at a median frequency of 12 x 10(-5) from individual ears from progeny of crosses between mouse strains 129/Sv and C3H/HeJ. The frequency of DAP(r) variants varied greatly among individual ears, suggesting that they preexisted in vivo and arose at various times during development. Polymorphic molecular markers and a cytological marker on the centromere of chromosome 8 made it possible to discriminate between each of six possible mechanistic pathways of LOH. The majority (about 80%) of the DAP(r) variants were a consequence of mitotic recombination. The prevalence of mitotic recombination in regions proximal to Aprt did not correlate with meiotic map distances. In particular, there was a higher than expected frequency of crossovers within the interval 59 cM to 67 cM. The high spontaneous frequency of Aprt LOH, mediated primarily by mitotic recombination, is fully consistent with our previous results with human peripheral T cells from individuals known to be heterozygous at APRT. Thus, this Aprt heterozygote mouse is a valid model for studying somatic mutagenesis and mitotic recombination in vivo.


Subject(s)
Adenine Phosphoribosyltransferase/genetics , Chromosome Mapping , Genetic Variation , Heterozygote , Loss of Heterozygosity , Recombination, Genetic , Adenine Phosphoribosyltransferase/deficiency , Animals , Crosses, Genetic , Ear, External , Exons , Female , Fibroblasts/enzymology , Genes, Recessive , Humans , Male , Mice , Mice, Inbred C3H , Mice, Inbred Strains , Mice, Mutant Strains , Mutagenesis , Point Mutation , Skin/cytology , Skin/enzymology , T-Lymphocytes/enzymology
19.
Am J Med Genet ; 86(2): 134-9, 1999 Sep 10.
Article in English | MEDLINE | ID: mdl-10449648

ABSTRACT

Cystinuria, a renal tubule disease affecting urinary cystine excretion with or without kidney stone formation, previously was mapped to chromosome region 2p.21. Mutations in the gene SLC3A1 or NBAT, the reported candidate gene for cystinuria at 2p.21, have been demonstrated in individuals with the autosomal recessive Type I cystinuria phenotype. Recently, the Type III cystinuria phenotype was mapped to chromosome region 19q13.1. Here we report a kindred of 39 persons in two families of cystinurics, Types II and III, that support linkage to 19q13.1 and exclude 2p.21. Based on a dominant model of inheritance, two-point analysis of the entire pedigree produced a maximum lod score (Z(max)) of 3.82 at marker D19S425. Multipoint analysis yielded a lod score of 4.96 at this marker, and a resultant lod score of 5.90 using a codominant model of inheritance. Furthermore, a candidate gene interval of 8.9 cM, flanked by markers D19S225 and D19S223, was obtained using multipoint and haplotype analyses. Thus, this kindred demonstrates the linkage of Type II cystinuria to 19q13.1 and confirms the linkage of Type III cystinuria at 19q13.1 while excluding the marker D19S225 that was previously included in the critical interval.


Subject(s)
Chromosomes, Human, Pair 19/genetics , Cystinuria/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Alleles , Child , Child, Preschool , Chromosome Banding , Family Health , Female , Genes, Dominant/genetics , Genetic Linkage , Genotype , Haplotypes , Humans , Lod Score , Male , Microsatellite Repeats , Middle Aged , Pedigree , Phenotype
20.
Exp Hematol ; 27(8): 1346-52, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10428512

ABSTRACT

Bone marrow transplantation is increasingly used as a treatment for numerous immunologic, hematologic, and malignant disorders. However, the mechanism by which transplanted hematopoietic stem cells are engrafted is not completely understood. Many traditional techniques have been used to study the engraftment of transplanted stem cells. Most of these methods are ex vivo and, in some cases, donor cells must be modified to enable detection. We describe a novel alternative for identifying unmodified primitive donor cells in a murine host. This mouse model is based on the differential capacity of adenine phosphoribosyltransferase (APRT)-positive and APRT-negative cells to sequester and incorporate radiolabeled adenine. Aprt is the gene encoding the adenine phosphoribosyltransferase purine salvage enzyme and has been ablated in 129sv mice. Following the injection of APRT-positive c-kit-positive enriched hematopoietic cells into syngeneic, sublethally irradiated APRT-deficient mice, engrafted cells and their presumptive progeny were successfully tracked by polymerase chain reaction. Their presence also was visualized by autoradiography of paraffin-embedded tissue sections. APRT-positive c-kit-positive enriched cells were detected in the bone marrow, spleen, lung, and thymus of nonirradiated mice. Donor cells and their progeny were more widely distributed in tissues of sublethally irradiated mice than of their nonirradiated counterparts, demonstrating that the pattern of localization of c-kit-positive enriched cells differs between nonirradiated and sublethally irradiated syngeneic recipients. The Aprt mouse model provides a sensitive method for further studying the mechanism of engraftment of unmodified donor hematopoietic cells in relation to the tissue architecture of the recipient.


Subject(s)
Adenine Phosphoribosyltransferase/metabolism , Adenine/pharmacokinetics , Carbon Radioisotopes/pharmacokinetics , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/diagnostic imaging , Adenine Phosphoribosyltransferase/genetics , Animals , Autoradiography , Cell Survival , Graft Survival , Hematopoietic Stem Cells/enzymology , Injections, Intravenous , Lymphoid Tissue/pathology , Metabolic Clearance Rate , Mice , Mice, Knockout , Paraffin Embedding , Polymerase Chain Reaction , Proto-Oncogene Proteins c-kit/analysis , Radiation Chimera , Radiation Injuries, Experimental/pathology , Radionuclide Imaging , Sensitivity and Specificity , Tissue Distribution
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