Genotoxic Stress Response Gene Expression in the Mid-Organogenesis Rat Conceptus

Robert K. Vinson and Barbara F. Hales1

Department of Pharmacology and Therapeutics, 3655 Promenade Sir William Osler, McGill University, Montréal, Québec, Canada H3G 1Y6

Received January 26, 2003; accepted March 25, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of the conceptus to respond to genotoxic stress may be critical for normal development, particularly after exposure to genotoxic teratogens. Members of the phosphatidylinositol 3-kinase (PI3K) superfamily are involved in controlling cell cycle activity and maintaining genomic stability. The expression of PI3K family members ATM, ATR, and DNA-PKcs, and downstream genes p53, GADD45, and p21, was examined in the mid organogenesis rat conceptus in vivo on gestational days (GD) 10 through 12 and in vitro following exposure to genotoxic stress. ATM was the most highly expressed PI3K family member in both yolk sac and embryo proper, with transcript levels increasing ~fourfold in the embryo from GD 10 to 12. Transcript concentrations for ATR, DNA-PKcs, and downstream genes were low in both tissues; all genes had increased transcript levels exclusively in the GD 12 embryo. Transient oxidative stress, induced by short-term, in vitro embryo culture, had no effect on transcript levels in either tissue. Culture for 24 or 44 h significantly decreased ATM transcript levels in both embryo and yolk sac, but downstream genes were unaffected compared to GD-11 and -12 in vivo levels, respectively. Exposure to 4-hydroperoxycyclophosphamide (4-OOHCPA), an activated form of the nitrogen mustard cyclophosphamide (CPA), had no effect on transcript levels for any of the genes examined. Therefore, while transcripts for genotoxic stress-response genes are present in the mid organogenesis rat conceptus, their expression is not regulated by exposure in culture to either transient oxidative stress or a genotoxic alkylating agent. The inability of the conceptus to upregulate transcripts in response to insult may contribute to an increased susceptibility to stressors during organogenesis.

Key Words: DNA damage; oxidative stress; cyclophosphamide; phosphatidylinositol 3-kinase; gene expression; cell cycle checkpoint.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability to sense and signal within the cell that DNA damage has occurred is one of the most important cellular defenses against genotoxic agents. DNA-damage sensors activate downstream cellular targets that delay the cell cycle, leading to checkpoint arrest (Lowndes and Murguia, 2000Go), modify DNA repair activity, and activate the apoptotic cascade (Shiloh, 2001Go). Members of the phosphatidylinositol 3-kinase (PI3K) superfamily are serine/threonine protein kinases that act as genotoxic stress sensors. This family includes ataxia-telangiectasia mutated (ATM; Savitsky et al., 1995Go), ATM- and Rad3-related (ATR; Cimprich et al., 1996Go), and the DNA-dependent protein kinase (DNA-PK; Lees-Miller et al., 1990Go). ATM is activated following ionizing radiation (IR; Canman and Lim, 1998Go) and oxidative stress (Rotman and Shiloh, 1997Go), ATR following UV radiation (UVR; Tibbetts et al., 1999Go) and exposure to alkylating agents such as cisplatin (Damia et al., 1998Go), and DNA-PK in response to double-strand breaks (DSBs; Smith and Jackson, 1999Go). Activation of ATM and ATR leads to the activation of p53 (Canman and Lim, 1998Go); activation of p53 transcriptionally upregulates p21 and the growth arrest and DNA damage-inducible gene 45 (GADD45; Wang et al., 1999Go). GADD45 triggers G2/M checkpoint arrest (Wang et al., 1999Go), while activation of p21 leads to G1/S checkpoint arrest (Sherr and Roberts, 1999Go), and can activate the apoptotic machinery (Dotto, 2000Go). DNA-PK, composed of a catalytic subunit, DNA-PKcs and the Ku70/80 heterodimer (Smith and Jackson, 1999Go), binds to DNA DSBs and activates the apoptotic machinery (Wang et al., 2000Go).

Both oxidative stress (Wells et al., 1997Go) and alkylating agents (Glantz, 1994Go) are teratogenic, inducing specific malformations during susceptible stages of development. The embryo culture system has been utilized as a model system to study these teratogens. Transient oxidative stress is induced within 30 min following the initiation of culture (Ozolins and Hales, 1997Go). Exposure of the conceptus to active metabolites of cyclophosphamide, a nitrogen mustard, is used to model the action of alkylating agents as teratogens (Slott and Hales, 1988Go). The rat conceptus is most sensitive to the effects of genotoxic teratogens during mid organogenesis, gestational day (GD) 10–12 (Jirakulsomchok and Yielding, 1984Go; Little and Mirkes, 1987Go; Platzek et al., 1982Go). Impaired responses to DNA damage may enhance the consequences of teratogen exposure and lead to specific birth defects. Previous studies have examined the gene expression profiles of the major DNA repair pathways in the mid organogenesis conceptus (Vinson and Hales, 2001aGo,bGo). Little is known about regulation of the expression of DNA damage-sensor genes during development. The present study was undertaken to elucidate the expression profiles of the PI3K superfamily genes during mid organogenesis in the rat conceptus, and to determine whether exposure to transient oxidative stress or to DNA damage with 4-hydroperoxycyclophosphamide (4-OOHCPA) alters the expression of these genes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue preparation.
Timed-pregnant virgin female Sprague-Dawley rats (225–250 g) were obtained from Charles River Canada (St. Constant, Québec, Canada) and housed in the McIntyre Animal Resource Centre (McGill University). Rat chow (Purina Rat Chow 5012, St. Louis, MO) and water were provided ad libitum, and animals were exposed to a 14-h light: 10-h dark cycle. All treatments were in accordance with a protocol approved by the Animal Care Committee of McGill University. Gestational-day zero was defined as the morning following mating. On GD 10, 11, and 12, uteri were removed and embryo and yolk sac tissues were dissected immediately, frozen individually in liquid nitrogen, and stored at -80°C.

Embryo culture.
Rat conceptuses were explanted from timed-pregnant dams on GD 10 and cultured using established techniques (New, 1978Go). The whole-embryo culture model removes any confounding maternal effects that may occur following drug exposure. For short-term oxidative stress studies, conceptuses with an intact yolk sac and ectoplacental cone were cultured for 0.5, 1.5, 3, and 6 h at 37°C in 90% heat-inactivated rat serum supplemented with penicillin and streptomycin. For 4-hydroperoxycyclophosphmide (4-OOHCPA) studies, conceptuses were cultured as above in the presence of vehicle (sterile water) or 10 µM 4-OOHCPA (a gift from M. Colvin) for either 24 or 44 h. In culture, 4-OOHCPA breaks down spontaneously in solution to its active metabolites (Slott and Hales, 1988Go), phosphoramide mustard and acrolein. Following culture, embryos were removed and dissected as described above.

Antisense RNA (aRNA) technique.
The aRNA technique was used to examine the DNA-repair gene-expression profile on a per embryo basis, as previously described (Vinson and Hales, 2001aGo,bGo). This technique is essentially a "reverse Northern blot," allowing for the simultaneous examination of multiple gene transcripts from a single tissue sample. Individual embryos and yolk sacs were sonicated on ice in lysis buffer (1 mg/ml digitonin, 5 mM DTT, 50 mM Tris pH 8.3, 6 mM MgCl2, 0.12 mM KCl), and reverse-transcribed for 2 h at 37°C in the presence of an oligo(dT) primer attached to a T7 RNA polymerase promoter, which recognizes and binds to the poly(A) tail of mRNA in the tissue sample. Self-priming allowed the formation of double-stranded cDNA by a combination of T4 DNA polymerase (Gibco BRL, Burlington, Ontario) and Klenow fragment (New England Biolabs, Mississauga, Ontario). Antisense RNA was created from the cDNA pool by T7 RNA polymerase (New England Biolabs) and simultaneously radiolabeled with a 32P CTP (10 mCi/mol; Amersham Pharmacia Biotech, Baie d’Urfé, Québec) for 4 h at 37°C. The linearity and reproducibility of this amplification reaction were determined by trichloroacetic acid precipitation of 32P CTP incorporated into the acid-insoluble fraction (data not shown).

Gene array membranes and hybridization.
To examine the expression of multiple genes using a single tissue sample, gene-expression arrays were created. In particular, the expression of six ATM pathway and family-member genes was examined. These were: ATM, ataxia-telangiectasia-related (ATR), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), growth arrest and DNA damage-inducible gene 45 (GADD45), p21, and p53. To create these arrays, nylon membranes (Zeta-Probe GT, Bio-Rad, Mississauga, Ontario) were slot-blotted (Bio-Dot SF, Bio-Rad) with equimolar amounts of cDNAs, as per the manufacturer’s protocol. Wherever possible, rodent cDNA probes were used. Nonrodent probes were chosen based on their high homology to rodent cDNAs for the genes of interest. Nonrodent probes were used for DNA-PKcs, p21, and ATR, all of which were human in origin. Gene expression arrays were prehybridized for 30 min in hybridization buffer (7% SDS, 0.12 M Na2HPO4 pH 7.2, 0.25 M NaCl, 50% formamide). Heat denatured, radiolabeled aRNA probes were hybridized overnight to arrays at 42°C.

Following hybridization, the arrays were washed in solutions of decreasing stringency (from 2x SSC/0.1% SDS to 0.1x SSC/0.1% SDS) at 42°C for 20 min each and exposed to phosphorimager plates overnight. Arrays were stripped of probe by boiling in 0.1x SSC/0.5% SDS twice for 20 min each. Stripping efficiency was determined by exposing membranes to phosphorimager plates overnight. Arrays were reused a maximum of five times, at which point no appreciable degradation in signal was observed (data not shown). Blots were initially randomly assigned to each sample group being studied; stripped blots were not reused for other individual samples from the same sample group. In addition, each sample group was tested on blots with, on average, the same number of stripping cycles, to minimize any chance of expression level bias owing to the stripping procedure.

Quantification and analysis of aRNA data.
Images of the gene expression arrays were obtained using a STORM phosphorimager (Molecular Dynamics, Sunnyvale, CA), and gene expression quantified using ImageQuant 5.0 software for Windows NT (Molecular Dynamics). Gene expression intensity values on each membrane were normalized relative to the expression of vimentin, a structural protein; vimentin was chosen because its expression remained constant between the different time points and tissues. The intensity value from pUC18 plasmid DNA blotted onto each array (nonspecific hybridization) was also subtracted from each gene intensity value prior to normalization to vimentin. In order to ensure the consistency and reliability of the data, several replicates for each tissue and treatment were performed; each embryo or yolk sac sample was extracted from tissues from separate litters. Data are from four separate GD 10–12 yolk sacs and embryo samples for each in vivo time point, from three to seven separate samples for culture-alone data per culture time point, and from three samples for culture with 4-OOHCPA data per culture time point, each obtained from separate litters.

Statistical analysis.
Statistical analysis was done on an individual-gene basis using one-way ANOVA, followed by either the Tukey post-hoc test or the Student’s t-test with SigmaStat version 2.03 software (SPSS, Chicago, IL).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of PI3K Superfamily and Downstream Stress-Response Genes during Organogenesis in Vivo
The expression patterns of PI3K family members, ATM, ATR, and DNA-PKcs in rat yolk sac and embryo-proper on GD 10, 11, and 12 are shown in Figure 1Go. ATM was the most highly expressed PI3K family gene examined in both tissues at all time points (Fig. 1AGo). ATM expression was similar in both tissues on GD 10 and 11; a dramatic fourfold increase in the steady-state concentrations of ATM transcripts occurred between GD 11 and 12, exclusively in the embryo. ATR and DNA-PKc were expressed at low levels in the yolk sac between GD 10 and 12 (Figs. 1BGo and 1CGo). The embryo-proper displayed transcript levels near the limit of detection for ATR and DNA-PKcs on GD 10, with higher transcript levels for both genes observed on GD 11 and 12 (Figs. 1BGo and 1CGo).



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FIG. 1. PI3K superfamily and downstream stress response gene expression during mid organogenesis in rat yolk sac (gray bars) and embryo proper (black bars): ATM (A), ATR (B), DNA-PKcs (C), p53 (D), GADD45 (E), and p21 (F). Values represent the mean transcript concentrations ± SEM relative to vimentin from four replicates. *Expression significantly different from GD 10, or **both GD 10 and GD 11 by one-way ANOVA and Tukey tests, p < 0.05.

 
The expression profiles for downstream genes, p53, GADD45, and p21, are shown in Figures 1DGo–1FGo. The expression of all three genes was low in both tissues. Transcript levels did not change significantly during organogenesis in the yolk sac; in the embryo, GADD45 and p21 transcript levels mirrored the increase seen for ATM, increasing on GD 12 compared to GD 10 (for GADD45; Fig. 1EGo) or compared to GD 10 and 11 (for p21; Fig. 1FGo).

Effect of Embryo Culture on PI3K Superfamily and Downstream Stress-Response Gene Expression
Embryo culture induces short-term oxidative stress (Ozolins and Hales, 1997Go). To determine if this oxidative stress affects PI3K superfamily gene expression, embryos were cultured for 0.5, 1.5, 3, and 6 h and the expression of PI3K superfamily and downstream genes was determined (Fig. 2Go). Short-term culture did not affect transcript concentrations of ATM (Fig. 2AGo), ATR (Fig. 2BGo), or DNA-PKcs (Fig. 2CGo), nor did it alter transcript levels for downstream targets p53, GADD45, and p21 (Figs. 2DGo–2FGo).



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FIG. 2. PI3K superfamily and downstream stress response-gene expression following short-term embryo culture in yolk sac (gray bars) and embryo proper (black bars): ATM (A), ATR (B), DNA-PKcs (C), p53 (D), GADD45 (E), and p21 (F). Values represent the mean transcript concentrations ± SEM relative to vimentin from three to seven replicates (n is specified on the top of each bar).

 
Embryos are cultured frequently for 24 or 44 h, in the presence and absence of putative teratogens, to elucidate the effects of these substances on organogenesis. To compare expression profiles of the ATM family of stress-response genes during organogenesis in vivo with those from embryos cultured in vitro, GD-10 embryos cultured for 24 h were compared to GD-11 embryos in vivo, while GD-10 embryos cultured for 44 h were compared to GD-12 embryos in vivo (Fig. 3Go). While a slight decrease in ATM transcript levels was observed in yolk sac following a 24-h culture compared to GD-11 in vivo values (Fig. 3AGo), a dramatic decrease to 15% of GD-12 levels was observed following 44 h of culture (Fig. 3BGo). p53 and GADD45 transcript levels were unaffected following either culture period (p21 levels were not examined). In the embryo, ATM transcript levels decreased to 22% of GD-11 (Fig. 3CGo), and 35% of GD-12 values (Fig. 3DGo), after 24- and 44-h culture, respectively. Apart from a decrease in GADD45 transcript levels to undetectable levels following a 44-h culture (Fig. 3DGo), p53 and GADD45 transcripts were unaffected by long-term culture.



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FIG. 3. ATM family stress response-gene expression in yolk sac (A, B) and embryo proper (C, D) following long-term embryo culture for 24 h (gray bars; A, C) and 44 h (gray bars; B, D) compared to GD 11 (dark hatched bars; A, C) or GD 12 in vivo (dark hatched bars; B, D). Values represent the mean transcript concentrations ± SEM, relative to vimentin from three to six replicates (n is specified on the top of each bar). *Expression significantly different from in vivo value by Student’s t-test, p < 0.05.

 
Effect of 4-OOHCPA on PI3K Superfamily and Downstream Stress-Response Gene Expression
Consequences of exposing GD-10 embryos to the alkylating agent and teratogen, 4-OOHCPA, on the expression of DNA damage, sensor genes are shown in Figure 4Go. Conceptuses were exposed to vehicle or drug (10 µM) in culture for 0.5, 1.5, 3, or 6 h (Fig. 4Go). No significant changes in ATM, ATR, or DNA-PKcs expression occurred following 4-OOHCPA exposure (Figs. 4AGo, 4BGo, and 4CGo). ATR and DNA-PKcs were expressed at or near the limit of detection in the embryo at most time points (Figs. 4BGo and 4CGo). Furthermore, p53, GADD45, and p21 transcript levels were unaffected by 4-OOHCPA exposure, in either yolk sac or embryo (Figs. 4DGo–4FGo).



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FIG. 4. PI3K superfamily and downstream stress-response-gene expression following short-term embryo culture with 10 µM 4-OOHCPA in yolk sac (gray bars) and embryo proper (black bars): ATM (A), ATR (B), DNA-PKcs (C), p53 (D), GADD45 (E), and p21 (F). Values represent the mean transcript concentrations ± SEM relative to vimentin from three replicates.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A key aspect in the response to DNA damage is coordination of the cell cycle to allow DNA repair to take place before DNA synthesis and cell division occur. The importance of PI3K family members for normal growth and development is apparent in the null-mutant animals (Friedberg and Meira, 2000Go). While some checkpoint functions may overlap between the PI3K family members, loss of more than one genotoxic stress sensor is not compatible with life; deficiencies in both ATM and other members of recombination and/or DNA damage sensor pathways are embryolethal, for example, ATM-null in DNA-PK null-mutant backgrounds (Friedberg and Meira, 2000Go). Null-mutant mice lacking downstream targets of PI3K family members also exhibit increased tumor susceptibility and checkpoint problems (Hollander et al., 2001Go; Vogelstein et al., 2000Go). We undertook analysis of the expression profile of these genes during a susceptible period of development to investigate the ability of the conceptus to respond to genotoxic teratogens.

Expression of PI3K Family Members and Downstream Genes in Vivo
PI3K family members are highly expressed during development in areas undergoing rapid cell division, consistent with a role for ATM in genome maintenance during cell division (Chen and Lee, 1996Go). ATM was the highest-expressed PI3K family member examined in both tissues. The dramatic increase in ATM transcript levels on GD 12 may be due to high levels of metabolic activity and DNA metabolism, both of which may increase the occurrence of internally generated DNA damage. While most studies have focused on regulation of ATM at the level of kinase activity, ATM transcript or protein levels have been shown as induced in several model systems in which cells are rapidly proliferating and/or differentiating (Clarke et al., 1998Go; Fukao et al., 1999Go). Interestingly, there was no increase in kinase activity as seen by Western blot analysis of phospho-(Ser/Thr) residues, which are substrates for both ATM and ATR (Vinson and Hales, unpublished data). ATM may be regulated either by alterations in the transcription of pre-existing ATM mRNA or by post-transcriptional mechanisms (Savitsky et al., 1997Go), possibly by specific proliferation-responsive signals within the rat conceptus.

Genes downstream of the ATM superfamily kinases were expressed at very low levels in the conceptus. Earlier studies found that p53 transcript levels are highest during organogenesis in the mouse (Rogel et al., 1985Go), with tissues undergoing differentiation exhibiting higher levels of p53 transcripts (Schmid et al., 1991Go). In the rat conceptus, a trend towards higher transcript levels appeared on GD 12, perhaps coinciding with differentiation of p53-dependent tissues. In this study, transcripts for both GADD45 and p21 appear between GD 11 and 12 in the embryo, mirroring ATM and DNA-PKcs transcript profiles, and in parallel to the expression profile for p53.

Transient Oxidative Stress and PI3K Pathway Gene Expression
Alteration of ATM expression may be beneficial during organogenesis to protect the embryo against increased oxidative stress as a result of high levels of metabolism or xenobiotic exposure (Wells et al., 1997Go). Culture elicits transient oxidative stress in the embryo, increasing oxidized glutathione and altering AP-1 transcription factor mRNA levels and DNA-binding activity (Ozolins and Hales, 1997Go). Oxidative stress did not affect ATM transcript levels. Nevertheless, long-term culture, for 24 or 44 h, did result in decreased ATM mRNA levels; this decrease was specific for ATM, as several other DNA repair-pathway genes were unaffected by culture conditions (data not shown). The explanation for this decrease is not clear; embryos cultured for 24 or 44 h have normal oxidized-to-reduced glutathione levels, indicative of a lack of oxidative stress at these time points (Ozolins and Hales, 1997Go). The decrease in ATM transcripts may be due to other stressors in the culture system such as the depletion of specific growth factors and other nutrients, which may lead to dysregulation of cell function. Alteration of ATM expression may be stressor-specific, or the response of the embryo to genotoxic stress may be unique. Limitations in the factors required for proper embryonic growth following extended periods of culture may play a role in the downregulation of stress-response genes.

Effect of 4-OOHCPA on PI3K Family Genes and Downstream Targets
Several DNA-repair pathways repair the damage caused by cyclophosphamide (CPA) (De Silva et al., 2000Go; McHugh et al., 2000Go). In particular, the nonhomologous end-joining (NHEJ) pathway is involved in repairing DSBs formed by nitrogen mustards (McHugh et al., 2000Go); DNA-PK and associated Ku subunits are involved in this recombination repair pathway (Lieber, 1999Go). Therefore, the expression of these genes may be crucial in repairing the genotoxic damage caused by CPA.

Previous studies have demonstrated a link between genotoxic stress and checkpoint arrest during organogenesis. Embryos cultured with 4-OOHCPA exhibit an accumulation of S-phase cells and G2/M checkpoint arrest, suggesting a relationship between cell cycle arrest and malformations (Little and Mirkes, 1992Go). CPA doses that are genotoxic but do not lead to malformations have been shown to perturb the cell cycle (Francis et al., 1990Go), suggesting that the embryo has the ability to repair genotoxic stress and, furthermore, that checkpoint arrest is integral in the prevention of teratogenesis. Perturbation of the cell cycle has been shown to occur approximately 5 h after CPA exposure (Little and Mirkes, 1992Go); it was hypothesized that the G2/M arrest was due to the presence of DNA cross-links that inhibit initiation of mitosis rather than an active arrest process. In this study, no changes in gene expression occurred near this time point for any PI3K family members or their downstream targets following exposure to 4-OOHCPA. If cell cycle arrest in the conceptus requires active transcription of these genes, then perhaps the conceptus is unable to activate these checkpoints, and the arrest seen is indeed due to genotoxic damage-induced structural abnormalities within the genome. The conceptal response may be stress-, tissue-, and developmental stage-specific, because exposure to methylmercury of rodent embryonic neuronal and limb-bud cells, in vitro, induced both cell-cycle arrest and an increase in GADD45 transcripts (Ou et al., 1997Go).

Apoptosis is a hallmark of the teratogenicity of CPA (Chen et al., 1994Go; Mirkes and Little, 1998Go). While CPA can induce DSBs, the role of PI3K family members in the response to these breaks is unclear. It is possible that PI3K family members play a role, both in the normal programmed cell death that occurs during development and in the aberrant cell death following teratogen exposure.

DNA-repair capability and downstream cellular responses to DNA damage are largely unknown during mammalian development. Only when the capacity of the conceptus to sense and respond to genotoxic teratogens is established, will the importance of these responses in the teratogenicity of genotoxic agents be determined. We suggest that one of the determinants of the ability of the conceptus to respond to oxidative stress and other genotoxic agents may be the expression of PI3K family members. The results of this study demonstrate that PI3K family members, as well as their downstream targets, are expressed in a time- and tissue-dependent manner in the rat conceptus during mid organogenesis and that they are not upregulated following genotoxic stress.


    ACKNOWLEDGMENTS
 
We thank the following individuals for the cDNAs provided for this study: Y. Shiloh for ATM, K. Cimprich for ATR, J.-P. Paiement for DNA-PKcs, T. Yoshida for GADD45, S. Benchimol for p53, and M. Bussemakers for vimentin. We also thank M. Colvin for providing 4-OOHCPA. This study was supported by the Canadian Institute of Health Research and FCAR Québec.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (514) 398-7120. E-mail: bhales{at}pharma.mcgill.ca. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Canman, C. E., and Lim, D. S. (1998). The role of ATM in DNA damage responses and cancer. Oncogene 17, 3301–3308.[CrossRef][ISI][Medline]

Chen, B., Cyr, D. G., and Hales, B. F. (1994). Role of apoptosis in mediating phosphoramide mustard-induced rat embryo malformations in vitro. Teratology 50, 1–12.[ISI][Medline]

Chen, G., and Lee, E. (1996). The product of the ATM gene is a 370-kDa nuclear phosphoprotein. J. Biol. Chem. 271, 33693–33697.[Abstract/Free Full Text]

Cimprich, K. A., Shin, T. B., Keith, C. T., and Schreiber, S. L. (1996). cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein. Proc. Natl. Acad. Sci. U.S.A. 93, 2850–2855.[Abstract/Free Full Text]

Clarke, R. A., Kairouz, R., Watters, D., Lavin, M. F., Kearsley, J. H., and Lee, C. S. (1998). Upregulation of ATM in sclerosing adenosis of the breast. Mol. Pathol. 51, 224–226.[Abstract]

Damia, G., Guidi, G., and D’Incalci, M. (1998). Expression of genes involved in nucleotide excision repair and sensitivity to cisplatin and melphalan in human cancer cell lines. Eur. J. Cancer 34, 1783–1788.[CrossRef][ISI][Medline]

De Silva, I. U., McHugh, P. J., Clingen, P. H., and Hartley, J. A. (2000). Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells. Mol. Cell. Biol. 20, 7980–7990.[Abstract/Free Full Text]

Dotto, G. P. (2000). p21WAF1/Cip1: More than a break to the cell cycle? Biochimica Biophysica Acta 1471, M43–M56.[CrossRef][ISI][Medline]

Francis, B. M., Rogers, J. M., Sulik, K. K., Alles, A. J., Elstein, K. H., Zucker, R. M., Massaro, E. J., Rosen, M. B., and Chernoff, N. (1990). Cyclophosphamide teratogenesis: Evidence for compensatory responses to induced cellular toxicity. Teratology 42, 473–482.[ISI][Medline]

Friedberg, E. C., and Meira, L. B. (2000). Database of mouse strains carrying targeted mutations in genes affecting cellular responses to DNA damage; Version 4. Mutat. Res. 459, 243–274.[ISI][Medline]

Fukao, T., Kaneko, H., Birrell, G., Gatei, M., Tashita, H., Yoshida, T., Cross, S., Kedar, P., Watters, D., Khana, K. K., et al. (1999). ATM is upregulated during the mitogenic response in peripheral blood mononuclear cells. Blood 94, 1998–2006.[Abstract/Free Full Text]

Glantz, J. C. (1994). Reproductive toxicology of alkylating agents. Obstet. Gynecol. Surv. 49, 709–715.[Medline]

Hollander, M. C., Kovalsky, O., Salvador, J. M., Kim, K. E., Patterson, A. D., Haines, D. C., and Fornace, A. J., Jr. (2001). Dimethylbenzanthracene carcinogenesis in Gadd45a-null mice is associated with decreased DNA repair and increased mutation frequency. Cancer Res. 61, 2487–2491.[Abstract/Free Full Text]

Jirakulsomchok, E., and Yielding, K. L. (1984). DNA damage and repair in mouse embryos following treatment transplacentally with methylnitrosourea and methylmethanesulfonate. Teratog. Carcinog. Mutagen. 4, 523–536.[ISI][Medline]

Lees-Miller, S. P., Chen, Y. R., and Anderson, C. W. (1990). Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. Mol. Cell. Biol. 10, 6472–6481.[ISI][Medline]

Lieber, M. R. (1999). The biochemistry and biological significance of nonhomologous DNA end-joining: an essential repair process in multicellular eukaryotes. Genes Cells. 4, 77–85.[Abstract/Free Full Text]

Little, S. A., and Mirkes, P. E. (1987). DNA cross-linking and single-strand breaks induced by teratogenic concentrations of 4-hydroperoxycyclophosphamide and phosphoramide mustard in postimplantation rat embryos. Cancer Res. 47, 5421–5426.[Abstract]

Little, S. A., and Mirkes, P. E. (1992). Effects of 4-hydroperoxycyclophosphamide (4-OOH-CP) and 4-hydroperoxydechlorocyclophosphamide (4-OOH-deClCP) on the cell cycle of post implantation rat embryos. Teratology 45, 163–173.[ISI][Medline]

Lowndes, N. F., and Murguia, J. R. (2000). Sensing and responding to DNA damage. Curr. Opin. Genet. Dev. 10, 17–25.[CrossRef][ISI][Medline]

McHugh, P. J., Sones, W. R., and Hartley, J. A. (2000). Repair of intermediate structures produced at DNA interstrand cross-links in Saccharomyces cerevisiae. Mol. Cell. Biol. 20, 3425–3433.[Abstract/Free Full Text]

Mirkes, P. E, and Little, S. A. (1998). Teratogen-induced cell death in post implantation mouse embryos: Differential tissue sensitivity and hallmarks of apoptosis. Teratology 5, 592–600.

New, D. A. T. (1978). Whole-embryo culture and the study of mammalian embryos during organogenesis. Biol. Rev. Camb. Philos. Soc. 53, 81–122.[ISI][Medline]

Ou, Y. C., Thompson, S. A., Kirchner, S. C., Kavanagh, T. J., and Faustman, E. M. (1997). Induction of growth arrest and DNA damage-inducible genes Gadd45 and Gadd153 in primary rodent embryonic cells following exposure to methylmercury. Toxicol. Appl. Pharmacol. 147, 31–38.[CrossRef][ISI][Medline]

Ozolins, T. R., and Hales, B. F. (1997). Oxidative stress regulates the expression and activity of transcription factor activator protein-1 in rat conceptus. J. Pharmacol. Exp. Ther. 280, 1085–1093.[Abstract/Free Full Text]

Platzek, T., Bochert, G., Schneider, W., and Neubert, D. (1982). Embryotoxicity induced by alkylating agents: 1. Ethylmethanesulfonate as a teratogen in mice. Arch. Toxicol. 51, 1–25.[CrossRef][ISI]

Rogel, A., Popliker, M., Webb, C. G., and Oren, M. (1985). p53 cellular tumor antigen: Analysis of mRNA levels in normal adult tissues, embryos, and tumors. Mol. Cell. Biol. 5, 2851–2855.[ISI][Medline]

Rotman, G., and Shiloh, Y. (1997). The ATM gene and protein: Possible roles in genome surveillance, checkpoint controls, and cellular defense against oxidative stress. Cancer Surveys 29, 285–304.[ISI][Medline]

Savitsky, K., Bar-Shira, A., Gilad, S., Rotman, G., Ziv, Y., Vanagaite, L., Tagle, D. A., Smith, S., Uziel, T., Sfez, S., et al. (1995). A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science 268, 1749–1753.[ISI][Medline]

Savitsky, K., Platzer, M., Uziel, T., Gilad, S., Sartiel, A., Rosenthal, A., Elroy-Stein, O., Shiloh, Y., and Rotman, G. (1997). Ataxia-telangiectasia: Structural diversity of untranslated sequences suggests complex post-translational regulation of ATM gene expression. Nucleic Acids Res. 25, 1678–1684.[Abstract/Free Full Text]

Schmid, P., Lorenz, A., Hameister, H., and Montenarh, M. (1991). Expression of p53 during mouse embryogenesis. Development 113, 857–865.[Abstract]

Sherr, C. J., and Roberts, J. M. (1999). CDK inhibitors: Positive and negative regulators of G1-phase progression. Genes Dev. 13, 1501–1512.[Free Full Text]

Shiloh, Y. (2001). ATM and ATR: Networking cellular responses to DNA damage. Curr. Opin. Genet. Dev. 11, 71–77.[CrossRef][ISI][Medline]

Slott, V. L., and Hales, B. F. (1988). Role of the 4-hydroxy intermediate in the in vitro embryotoxicity of cyclophosphamide and dechlorocyclophosphamide. Toxicol. Appl. Pharmacol. 92, 170–178.[CrossRef][ISI][Medline]

Smith, G. C. M., and Jackson, S. P. (1999). The DNA-dependent protein kinase. Genes Dev. 13, 916–934.[Free Full Text]

Tibbetts, R. S., Brumbaugh, K. M., Williams, J. M., Sarkaria, J. N., Cliby, W. A., Shieh, S.-Y., Taya, Y., Prives, C., and Abraham, R. T. (1999). A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev. 13, 152–157.[Abstract/Free Full Text]

Vinson, R. K., and Hales, B. F. (2001a). Expression of base excision, mismatch, and recombination repair genes in the organogenesis-stage rat conceptus and effects of exposure to a genotoxic teratogen, 4-hydroperoxycyclophosphamide. Teratology 64, 283–291.[ISI][Medline]

Vinson, R. K., and Hales, B. F. (2001b). Nucleotide excision repair gene expression in the rat conceptus during organogenesis. Mutat. Res. 486, 113–123.[ISI][Medline]

Vogelstein, B., Lane, D., and Levine, A. J. (2000). Surfing the p53 network. Nature 408, 307–310.[CrossRef][ISI][Medline]

Wang, S., Guo, M., Ouyang, H., Li, X., Cordon-Cardo, C., Kurimasa, A., Chen, D. J., Fuks, Z., Ling, C. C., and Li, G. C. (2000). The catalytic subunit of DNA-dependent protein kinase selectively regulates p53-dependent apoptosis but not cell-cycle arrest. Proc. Natl. Acad. Sci. U.S.A. 97, 1584–1588.[Abstract/Free Full Text]

Wang, X. W., Zhan, Q., Coursen, J. D., Khan, M. A., Kontny, H. U., Yu, L., Hollander, M. C., O’Connor, P. M., Fornace, A. J., Jr., and Harris, C. C. (1999). GADD45 induction of a G2/M cell cycle checkpoint. Proc. Natl. Acad. Sci. U.S.A. 96, 3706–3711.[Abstract/Free Full Text]

Wells, P. G., Kim, P. M., Laposa, R. R., Nicol, C. J., Parman, T., and Winn, L. M. (1997). Oxidative damage in chemical teratogenesis. Mutat. Res. 396, 65–78.[CrossRef][ISI][Medline]





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