-stimulating of (ASPP2) heterozygous mice are tumor-prone and have attenuated cellular damage–response thresholds

Kerstin M. Kampaa,b, Jared D. Acobaa, Dexi Chena, Joel Gaya, Hunjoo Leea, Kelly Beemera, Emerson Padiernosa, Nataya Boonmarkc, Zhiyi Zhua, Alice C. Fanc, Alexis S. Baileya,d, William H. Fleminga,d, Christopher Corlessa, Dean W. Felsherc, Louie Naumovskic,1, and Charles D. Lopeza,2

aDepartment of Medicine, Division of Hematology and Medical Oncology, Oregon Health and Science University, Portland, OR 97239; bMedizinsche Universita¨tsklinik, Department of Hematology, Oncology, Rheumatology, Immunology, and Pulmology, Universita¨t Tu¨ bingen, Tu¨bingen 72076, Germany; cDepartments of Medicine and Pediatrics, Division of Hematology and Oncology, Stanford University, Stanford, CA 94305; and dOregon Stem Cell Center, Portland, OR 97239

Edited by Brian J. Druker, Oregon Health and Science University, Portland, OR, and approved December 30, 2008 (received for review September 11, 2008) The expression of ASPP2 (53BP2L), a proapoptotic member of a In this report, we targeted the ASPP2 allele in a mouse by family of p53-binding , is frequently suppressed in many using homologous recombination to explore the in vivo con- human . Accumulating evidence suggests that ASPP2 inhib- sequences of attenuated ASPP2 expression. We demonstrate its tumor growth; however, the mechanisms by which ASPP2 that reduced ASPP2 expression in heterozygous mice results suppresses tumor formation remain to be clarified. To study this, in: (i) an increased incidence of a variety of spontaneous we targeted the ASPP2 allele in a mouse by replacing exons 10–17 tumors, (ii) the accelerated formation of high-grade thymic T -with a neoR . ASPP2؊/؊ mice were not viable because of an cell lymphomas in ␥-irradiated mice, and (iii) after ␥-irradia early embryonic lethal event. Although ASPP2؉/؊ mice appeared tion, an attenuated apoptotic response in ASPP2ϩ/Ϫ primary ϩ/Ϫ developmentally normal, they displayed an increased incidence of thymocytes and an attenuated G0/G1 checkpoint in ASPP2 a variety of spontaneous tumors as they aged. Moreover, ␥-irra- primary mouse embryonic fibroblasts (MEFs). These data diated 6-week-old ASPP2؉/؊ mice developed an increased inci- provide significant insight into the observation that ASPP2 dence of high-grade T cell lymphomas of thymic origin compared expression is reduced in human cancers, and suggest a mech- with ASPP2؉/؉ mice. Primary thymocytes derived from ASPP2؉/؊ anism by which disruption of ASPP2 pathways may play a role mice exhibited an attenuated apoptotic response to ␥-irradiation in tumorigenesis and response to therapy. -compared with ASPP2؉/؉ thymocytes. Additionally, ASPP2؉/؊ pri mary mouse embryonic fibroblasts demonstrated a defective G0/G1 Results checkpoint after ␥-irradiation. Our results demonstrate Generation of ASPP2؉/؊ Mice. To rigorously test the hypothesis that ASPP2 is a haploinsufficient tumor suppressor and, impor- that ASPP2 may have a tumor suppressor function, we targeted tantly, open new avenues for investigation into the mechanisms by ASPP2 in a mouse by using homologous recombination (Fig. which disruption of ASPP2 pathways could play a role in tumori- 1). The targeting vector was designed to disrupt exons 10–17, genesis and response to therapy. which also include codons for the repeat and SH3 domain required for interaction with p53 family members poptosis-stimulating protein of p53-2 (ASPP2), also known (exons 14–17; Fig. 1A). The targeting vector was electropo- Aas 53BP2L, encoded by TP53BP2 (1–3), enhances dam- rated into 129/SvJ ES cells, and clones were derived by age-induced apoptosis at least in part through a p53-mediated positive–negative selection in G418/gancyclovir. Approxi- pathway (2, 4–6). Depending on cell context and type of stress, mately 250 clones were screened to identify 4 positive clones ASPP2 levels increase via transcriptional or posttranslational by Southern blot analysis (Fig. 1B). Two separate clones were mechanisms after cellular damage (4, 6). In addition to expanded and injected into C57BL/6 blastocysts to generate interacting with p53 (and family members) (5, 7), ASPP2 several highly chimeric mice. Chimeras were crossed with protein, and the 123-aa, amino-terminal, truncated splice C57BL/6 mice, and offspring were screened with 2 separate isoform 53BP2/Bbp, also known as 53BP2S (3), interacts with PCR strategies and further confirmed by Southern blot anal- several proteins involved in modulating apoptosis and cell ysis (Fig. 1 B and C). Western blot analysis using 2 different growth, including Bcl-2, p65/RelA subunit of NF-␬B, Yes- antibodies recognizing the amino terminus (anti-ASPP2 Ab1) associated protein-1, HCV core protein, APCL, and protein or the carboxy terminus (anti-ASPP2 Ab2) demonstrated an phosphatase-1 (8–13). Additionally, ASPP2 is a direct E2F approximate 2-fold reduction in ASPP2 protein levels in target gene, suggesting that it is a common link between the ASPP2ϩ/Ϫ MEFs, thymus, and liver (Fig. 1D). Rb/E2F and p53/ pathways (14–16). ASPP2 expression is suppressed in many human cancers, and it has been associated with poor clinical outcome in patients with aggressive non- Author contributions: K.M.K., J.D.A., D.C., J.G., H.L., N.B., A.C.F., W.H.F., L.N., and C.D.L. designed research; K.M.K., J.D.A., D.C., J.G., H.L., K.B., E.P., N.B., Z.Z., A.C.F., A.S.B., and Hodgkin’s lymphoma treated with chemotherapy (2, 17–24). C.D.L. performed research; D.W.F. contributed new reagents/analytic tools; K.M.K., J.D.A., These findings suggest that ASPP2 is involved in important D.C., J.G., H.L., N.B., Z.Z., A.C.F., W.H.F., C.C., D.W.F., L.N., and C.D.L. analyzed data; and tumor suppression networks and the cellular damage response. K.M.K. and C.D.L. wrote the paper. Overexpression of ASPP2 or Bbp/53BP2S can suppress E1A The authors declare no conflict of interest. and ras-mediated transformation of rat embryo fibroblasts (25, This article is a PNAS Direct Submission. 26), whereas attenuation of ASPP2 expression promotes clo- 1Present address: Genentech Inc., 1 DNA Way, South San Francisco, CA 94080. nogenic survival and inhibits apoptosis in cell culture (2, 4, 6) 2To whom correspondence should be addressed. E-mail: [email protected]. and promotes tumor formation in vivo (27). However, the This article contains supporting information online at www.pnas.org/cgi/content/full/ mechanisms by which reduced ASPP2 expression enhances 0809080106/DCSupplemental. tumor formation in vivo remain to be elucidated. © 2009 by The National Academy of Sciences of the USA

4390–4395 ͉ PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809080106 Downloaded by guest on September 23, 2021 Fig. 1. Generation of ASPP2ϩ/Ϫ mice. (A) Schema of ASPP2 allele and targeting vector. Black boxes indicate coding exons; white boxes indicate untranslated regions. Neo indicates neomycin resistance gene; TK indicates thymidine kinase gene. (B) Strategies for detecting a wild-type ASPP2 allele (Upper) or integration of the targeting vector (Lower). Probe locations for Southern blot analysis are shown as gray bars. Open arrows indicate PCR set 1 primers. Neo-PCR indicates NeoR gene amplicon. WT-PCR indicates ASPP2 exon 13 amplicon. (C) Genotyping using PCR (Upper) and Southern blot analysis (Lower). (D) Western blots on equivalent amounts of total protein from ASPP2ϩ/Ϫ and ASPP2ϩ/ϩ MEFs (Upper), or thymuses and livers (Lower), using anti-ASPP2 Ab1 or anti-ASPP2 Ab2. Fold-expression (relative to ϩ/ϩ) normalized to tubulin.

ASPP2؊/؊ Mice Are Not Viable Because of an Embryonic Lethal Event. expression in available tumors arising in heterozygous mice did Although ASPP2ϩ/Ϫ mice appeared normal and reproduced, we not reveal loss of expression by quantitative RT-PCR (Fig. S1) could not identify viable ASPP2Ϫ/Ϫ pups. Genotyping of new- or by Western blot analysis (Fig. S2). To further explore whether born litters demonstrated an increased frequency of ASPP2ϩ/ϩ ASPP2 cooperated with p53 to suppress tumor development, we and ASPP2ϩ/Ϫ pups over the expected frequencies, consistent generated ASPP2ϩ/Ϫ;p53ϩ/Ϫ and ASPP2ϩ/ϩ;p53ϩ/Ϫ mice and with an embryonic lethal defect (Fig. 2). We performed timed determined the incidence of tumor formation as they aged. harvests as early as embryonic day 6.5 but were unsuccessful in Although, as expected (28), loss of a p53 allele increased the characterizing ASPP2Ϫ/Ϫ embryos. To explore whether the incidence of spontaneous tumors, we found that an ASPP2ϩ/Ϫ lethal ASPP2Ϫ/Ϫ phenotype could be altered, we generated background did not further accelerate tumorigenesis in p53ϩ/Ϫ ASPP2ϩ/Ϫ;p53ϩ/Ϫ mice and intercrossed them. However, we mice (Fig. S3). could not produce ASPP2Ϫ/Ϫ mice or embryos, regardless of the background p53 genotype, nor could we produce them in an ASPP2؉/؊ Mice Have an Increased Incidence of ␥-Irradiation-Induced inbred BALB/c background. High-Grade Lymphomas. Because ASPP2 is damage-inducible (2, 4, 6) and has a tumor-suppression function (Fig. 3), we reasoned ϩ Ϫ ASPP2؉/؊ Mice Have an Increased Incidence of Spontaneous Tumors. that ASPP2 / mice would demonstrate an increased incidence MEDICAL SCIENCES Because human cancers can have reduced ASPP2 levels (2, of ␥-irradiation-induced tumors. To examine this, we ␥-irradi- 17–24), we determined the spontaneous tumor-free survival of ated 6-week-old ASPP2ϩ/ϩ and ASPP2ϩ/Ϫ mice with a total of 6.0 ASPP2ϩ/Ϫ mice over an extended period (Fig. 3A). ASPP2ϩ/ϩ Gy or 10.5 Gy (in divided weekly fractions) and measured the mice demonstrated the expected incidence of spontaneous tu- tumor-free survival (Fig. 4A). ASPP2ϩ/ϩ mice did not develop mors seen in mice of similar background strain (28). In contrast, tumors during the observed time. However, ASPP2ϩ/Ϫ mice ASPP2ϩ/Ϫ mice had a significant increase in tumor formation developed tumors after 6.0-Gy or 10.5-Gy irradiation (P ϭ 0.024 (P ϭ 0.011, log-rank test). There was a similar spectrum of tumor and P ϭ 0.045 respectively, log-rank test). In the ASPP2ϩ/Ϫ mice types in both genotypes (Fig. 3B). Examination of ASPP2 that developed tumors, histopathology demonstrated aggressive

Kampa et al. PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 ͉ 4391 Downloaded by guest on September 23, 2021 Fig. 2. ASPP2Ϫ/Ϫ mice are not viable. Indicated genotypes of newborn pups from ASPP2ϩ/Ϫ matings are shown. The expected Mendelian frequencies are indicated by black columns; observed ASPP2 genotypes are indicated by white columns.

high-grade lymphomas, along with circulating lymphoma cells in the peripheral blood and bone marrow (Fig. 5). Immunopheno- typing of bone marrow typically demonstrated near-complete replacement by tumor cells that had a light scatter profile characteristic of large lymphocytes and expressed the panhema- topoietic marker CD45. As shown in Fig. 5B, tumor cells also coexpressed T cell markers, including CD5 and CD8, but did not

Fig. 4. ASPP2ϩ/Ϫ mice have an increased incidence of ␥-irradiation-induced lymphomas. (A) Kaplan–Meier lymphoma-free survival curves for ASPP2ϩ/ϩ and ASPP2ϩ/Ϫ mice after 6.0-Gy or 10.5-Gy (P ϭ 0.024 or P ϭ 0.045, log-rank test) total ␥-irradiation delivered in divided weekly fractions starting at 6 weeks old. (B) Kaplan–Meier lymphoma-free survival curves for tet-o- MYC;E␮SR-tTA mice (29) after conditional c-Myc expression in an ASPP2ϩ/ϩ or ASPP2ϩ/Ϫ background (P ϭ 0.97, log-rank test).

express CD4 or Nk1.1 (natural killer cell), B cell (B220), and myeloid (Mac-1/Gr-1) markers, nor did they express progenitors (c-kit) (Fig. S4). These results are consistent with a high-grade T cell lymphoma of thymic origin. To explore whether oncogene-induced T cell lymphomas would be accelerated in ASPP2 haploinsufficient mice, we crossed our ASPP2ϩ/Ϫ mouse with a transgenic mouse that develops T cell lymphomas after conditional expression of the ϩ/Ϫ Fig. 3. ASPP2 mice have an increased incidence of spontaneous tumors. MYC protooncogene in hematopoietic cells (29). These mice (A) Kaplan–Meier tumor-free survival curves for ASPP2ϩ/ϩ and ASPP2ϩ/Ϫ mice (P ϭ 0.011, log-rank test). (B) H&E-stained microscopic sections of represen- contain both a MYC cDNA under the control of a tetracycline- tative tumors found in ASPP2ϩ/Ϫ mice and a graph of tumor type frequency responsive minimal promotor (tet-o-MYC), and a tetracycline- between genotypes (P ϭ n.s., Fisher exact test). transactivating protein (tTA) under control of the Ig heavy-chain

4392 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809080106 Kampa et al. Downloaded by guest on September 23, 2021 Fig. 5. High-grade thymic T cell lymphomas induced in ASPP2ϩ/Ϫ mice after ␥-irradiation. (A) Thymic lymphoma (red arrow) in an ASPP2ϩ/Ϫ mouse. H&E- stained microscopic sections of lymphoma-infiltrated liver, kidney, and lung from this mouse. (B) Immunophenotyping of tumor cells in unfractionated bone marrow from this mouse. A total of 2% of normal myeloid progenitors coexpressed Mac-1 and Gr-1 (Upper Right). A total of 98% of the bone marrow was replaced by tumor cells expressing CD5 and CD8 (Lower), but not B220 (Upper Left), CD4, and Nk1.1 markers (Lower).

enhancer and the SR␣ (E␮SR-tTA). MYC transgene expression was induced by doxycycline withdrawal, and tumor- ϩ Ϫ free survival was determined. No significant difference was Fig. 6. ASPP2 / thymocytes have an attenuated apoptotic response to ␥ ϩ/ϩ ϩ/Ϫ observed in T cell lymphoma development between the -irradiation. Flow cytometry on ASPP2 or ASPP2 primary thymocytes 4 h after 5-Gy ␥-irradiation (right column) or 0-Gy ␥-irradiation (left column). The ϩ/ϩ ϩ/Ϫ ASPP2 and ASPP2 backgrounds (Fig. 4B). percentage of cells in each quadrant (relative to total cells) is indicated. The fold-increase in apoptosis between 0-Gy and 5-Gy ␥-irradiated thymocytes was ؉ ؊ ASPP2 / Thymocytes Have an Attenuated Apoptotic Response to determined by the increase in percentage of Annexin V-stained cells (lower ␥-Irradiation. Because ASPP2ϩ/Ϫ mice are predisposed to ␥-irra- right plus upper right quadrants). diation-induced thymic lymphomas (Figs. 4 and 5), we wanted to determine whether ASPP2ϩ/Ϫ thymocytes had an attenuated 1.9-, 1.4-, and 2.0-fold increases in the percentage of Annexin apoptotic response to ␥-irradiation. Primary thymocytes were ϩ ϩ ϩ Ϫ V-staining cells after ␥-irradiation. isolated from sibling ASPP2 / and ASPP2 / mice, maintained ␥ in short-term culture for 24 h, subjected to 5-Gy -irradiation, ؉/؊ ASPP2 MEFs Have an Attenuated G0/G1 Cell Cycle Checkpoint After and then assayed 4 h later (a dose and time point that induces ␥ Ͻ -Irradiation. To explore how reduced ASPP2 expression might 50% apoptosis). The fold-change in the percentage of apo- affect nonapoptotic cellular stress response pathways, we used ptotic cells was determined by flow cytometry and Annexin- primary MEFs (which normally undergo a ␥-irradiation-induced propidium iodide (PI) staining on irradiated thymocytes or on G0/G1 arrest) to measure changes in cell cycle checkpoints 24 h ϩ ϩ nonirradiated control thymocytes cultured in parallel (Fig. 6). after5Gyof␥-irradiation (Fig. 7). As expected, ASPP2 / MEDICAL SCIENCES Dotplots from 3 separate sets of paired sibling ASPP2ϩ/ϩ and ϩ Ϫ MEFs had a significant increase in the percentage of cells in ASPP2 / thymocytes are shown with (Fig. 6, right column) and G /G (from 57% to 75%; Fig. 7 Left). In contrast, ␥-irradiated ϩ ϩ 0 1 without (Fig. 6, left column) ␥-irradiation. In the ASPP2 / ASPP2ϩ/Ϫ MEFs did not have a significant increase in the thymocytes (Fig. 6, top rows of paired sets), there were 3.4-, 2.8-, percentage of cells in G0/G1 (Fig. 7 Right). and 3.1-fold increases in the percentage of Annexin V-staining cells (Fig. 6, lower right and upper right quadrants) after Discussion ␥-irradiation. In contrast, in the matched sibling ASPP2ϩ/Ϫ We found that aging ASPP2ϩ/Ϫ mice have accelerated sponta- thymocytes (Fig. 6, bottom rows of paired sets), there were only neous tumor development compared with ASPP2ϩ/ϩ mice (Fig.

Kampa et al. PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 ͉ 4393 Downloaded by guest on September 23, 2021 We did not observe genetic cooperation between ASPP2 and p53 to suppress tumor development (Fig. S3). This is partially consistent with the recent report on another ASPP2ϩ/Ϫ;p53ϩ/Ϫ mouse showing ASPP2 does not cooperate with p53 to suppress sarcoma or lymphoma development at 72 weeks, although at 42 weeks there is an increase in lymphomas (27, 30). Given that ASPP2 and p53 cooperation may be tumor type-specific (27), subtle strain-specific modifiers and/or the different targeting strategies could account for the differences. It is also possible that our ASPP2ϩ/Ϫ;p53ϩ/Ϫ mouse experiment was underpowered to detect modest alterations in tumor latency. Thus, we cannot firmly conclude that ASPP2 tumor suppression is p53- independent, although this raises the intriguing possibility that p53-independent mechanisms are involved (5, 8–16). Likewise, ASPP1 does not cooperate with p53 in vivo, because impaired Ϫ/Ϫ ϩ Ϫ lymphatic development of ASPP1 mice is not altered in a Fig. 7. ASPP2 / MEFs have an attenuated G /G checkpoint. Cell cycle 0 1 ϩ/Ϫ Ϫ/Ϫ ϩ/ϩ p53 or p53 background (36). distribution shows an increase in G0/G1-arrested ASPP2 MEFs but not ϩ/Ϫ ASPP2ϩ/Ϫ MEFs 24 h after 5-Gy ␥-irradiation (P ϭ 0.0052 and P ϭ 0.9, unpaired The attenuated apoptotic response of primary ASPP2 2-tailed Student’s t test, respectively). Mean values of triplicate experiments thymocytes (Fig. 6) suggests a potential mechanism for lym- are shown with SDs. phomagensis in ␥-irradiated mice (Figs. 4 and 5), because apoptotic defects may result in the persistence of thymocytes that have acquired tumorigenic mutations. Interestingly, we found a 3A). The large number of mice in this experiment strengthens ϩ Ϫ G /G checkpoint defect in ASPP2 / MEFs (Fig. 7). Because our finding that ASPP2 is a haploinsufficient tumor suppressor. 0 1 tumor suppression involves a variety of cellular functions in Consistent with other reports (27, 30), we did not find ASPP2 loss ϩ Ϫ addition to apoptosis (34, 37), this tantalizingly suggests that of heterozygosity in tumors arising in ASPP2 / mice (Figs. S1 ASPP2 function is far more complex than just simply enhancing and S2). Because ASPP2 promotes damage-induced apoptosis and proapoptotic p53 transcriptional programs (2). Previous reports inhibits survival in cell culture (2, 4–6), our findings that using tumor cell culture have also hinted that ASPP2 (and/or ASPP2ϩ/Ϫ mice have a significant increase in ␥-irradiation- isoforms) might modulate expression of cell cycle-regulating induced T cell lymphomas provides in vivo evidence that ASPP2 (25), perturb cell cycle progression (9), bind and modulate plays a role in damage-induced tumor suppression in susceptible other proteins (8–13, 38, 39), or be regulated by pathways organs (Fig. 4A). However, at higher doses (12 Gy), this was not involved in diverse functions (6, 14–16). The net result of these statistically significant, as might be expected if the genotoxic G0/G1 checkpoint (or other) defects in ASPP2 haploinsufficient damage exceeded a threshold in which reduced ASPP2 levels cells could manifest as a tumor-prone phenotype because of the could not accelerate lymphomagenesis. Interestingly, reduced accumulation of cell populations that will ultimately evolve into ASPP2 levels did not accelerate development of Myc-induced frank neoplastic growth. The molecular mechanisms underlying ϩ Ϫ T cell lymphomas (Fig. 4B), even though defects in p53 can the apoptotic and cell cycle defects in ASPP2 / cells remain accelerate lymphomas in this model (31). Perhaps ASPP2 has unknown, and we are currently exploring how reduced ASPP2 a more important role in tumor suppression after genotoxic expression can modulate global transcriptional and posttran- damage-induced (as opposed to oncogene-induced) tumori- scriptional networks. genesis, suggesting a role in DNA repair and maintenance of Our findings provide in vivo evidence that reduction of ASPP2 genomic stability. However, the oncogenic context driving expression results in acceleration of spontaneous and ␥-irradi- tumor formation—along with defective tumor suppressor ation-induced tumors, and that apoptotic and cell cycle arrest pathways—is incompletely understood (refs. 32–34 and refer- damage checkpoints are attenuated. These results rigorously ences within), and ASPP2 function in these different contexts confirm the mounting evidence for the role of ASPP2 as a tumor remains to be clarified. Moreover, high levels of MYC trans- suppressor (18–24, 27), and they expand upon potential mech- in this mouse (29) may overwhelm modest anisms of how ASPP2 functions. tumor suppression defects in ASPP2ϩ/Ϫ mice; thus, we cannot rule out that ASPP2 could suppress MYC-induced lymphomas Methods in other model systems. ASPP2 Allele-Targeted Disruption. ASPP2ϩ/Ϫ mice were generated by using the Ϫ Ϫ ASPP2 / mice die from an unknown early embryonic lethal Oregon Health and Science University (OHSU) Transgenic Core. Details are in defect (Figs. 1 and 2) that could not be compensated for by the SI Materials and Methods. homologous proapoptotic family member ASPP1 (2). Although in vitro evidence has not shown major functional differences Quantitative RT-PCR. Reverse transcription was performed on total RNA and between ASPP2 and ASPP1 (2, 5, 35), the nonredundant in vivo quantitative PCR was performed with TaqMan technology (Invitrogen) using findings suggest that ASPP2 has distinct biologic functions. standard conditions described in ref. 19. Details are in SI Materials and Conversely, the defective lymphatic vessel formation of Methods. ASPP1Ϫ/Ϫ mice suggests ASPP1 has distinct in vivo functions Western Blotting. Western blot analysis was performed as described in ref. 4 that cannot be compensated by ASPP2 (36). In contrast to our and in SI Materials and Methods. Rabbit anti-ASPP2 Ab1 was raised against an findings, another ASPP2-deficient mouse exhibits early postnatal expressed GST-amino terminus ASPP2 fusion protein (gift from Rachael Neve, lethality (27). Although background strain may contribute to Harvard University, Boston, MA) (40); rabbit anti-ASPP2 Ab2 was as described differences in developmental phenotypes, it is likely that the in ref. 9. different targeting strategies to disrupt exon 3 (27) or exons 10–17 (Fig. 1) may also contribute. Indeed, the complexity of the Cell Culture. Details are in SI Materials and Methods. Day 11.5 MEFs were ASPP2 locus suggests there might be multiple gene products, and prepared using standard techniques (41). Thymocytes were isolated from 5- to we are investigating this hypothesis. 6-week-old mice and cultured for Ͻ24 h before use.

4394 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809080106 Kampa et al. Downloaded by guest on September 23, 2021 Flow Cytometry. Flow cytometry was performed by using standard techniques leukemia/lymphoma cells, were killed for necropsy. Expert hematopathologic with details in SI Materials and Methods. Apoptosis by Annexin V staining (6), review was provided by the OHSU Pathology Core. Survival curves were cell cycle analysis by PI staining (14), and tumor cell imunophenotyping (42) calculated with SPSS v15.0 software. were all performed as described previously. ACKNOWLEDGMENTS. This work was supported in part by U.S. Public Health Mouse Colonies and Tumor Monitoring. IUCAC-compliant protocols were ap- Service Grants CA104997 and CA85773 (to C.D.L.), CA076316 (to L.N.), proved by the OHSU Department of Comparative Medicine. Details are in SI HL069133 and HL077818 (to W.H.F.), CA89305 (to D.W.F.), 5-P30-CA69533 (to Materials and Methods. Newly weaned pups were irradiated with weekly 3-Gy the OHSU Knight Institute), P01 CA034233 (to the Stanford University fractions (6 Gy total dose) or weekly 2.62-Gy fractions (10.5 Gy total dose). Lymphoma Program Project Grant); the Burroughs Welcome Fund, the Damon Starting at 10 weeks after irradiation, mice were monitored weekly for tumors Runyon Foundation (to D.W.F.), the Leukemia and Lymphoma Society (to and automated complete cell counts. Moribund mice, or mice with circulating A.C.F.), and gifts in the memory of Dr. Donald Peterson (to C.D.L.).

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