Cancer Resistance in the Blind Mole Rat Is Mediated by Concerted Necrotic Cell Death Mechanism

Cancer resistance in the blind mole rat is mediated by concerted necrotic cell death mechanism

Vera Gorbunovaa,1, Christopher Hinea,2, Xiao Tiana, Julia Ablaevaa, Andrei V. Gudkovb, Eviatar Nevoc,1, and Andrei Seluanova,1

aDepartment of Biology, University of Rochester, Rochester, NY 14627; bDepartment of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY 14263; and cInstitute of Evolution, University of Haifa, Haifa 31905, Israel

Contributed by Eviatar Nevo, October 3, 2012 (sent for review August 28, 2012) Blind mole rats Spalax (BMR) are small subterranean rodents com- tumors (12). The R174K substitution affects the DNA-binding mon in the Middle East. BMR is distinguished by its adaptations to domain of p53; the resulting protein is capable of inducing cell- life underground, remarkable longevity (with a maximum docu- cycle arrest but is defective in initiating apoptosis. We hypoth- mented lifespan of 21 y), and resistance to cancer. Spontaneous esized that R174K substitution evolved in BMR as an adaptation tumors have never been observed in spalacids. To understand the to the hypoxic environment in underground tunnels to prevent mechanisms responsible for this resistance, we examined the hypoxia-induced apoptosis (12). Mice with the corresponding growth of BMR fibroblasts in vitro of the species Spalax judaei p53 mutation are defective in p53-dependent apoptosis (13). − − and Spalax golani. BMR cells proliferated actively for 7–20 popu- These mice show longer tumor latency than p53 / mice, but are lation doublings, after which the cells began secreting IFN-β, and more tumor-prone than the wild-type mice (13). Thus, the ex- the cultures underwent massive necrotic cell death within 3 d. The treme tumor resistance of BMR is intriguing. necrotic cell death phenomenon was independent of culture con- Our previous studies of another long-lived subterranean rodent, ditions or telomere shortening. Interestingly, this cell behavior the naked mole rat (Heterocaphalus glaber), identified a novel was distinct from that observed in another long-lived and can- anticancer mechanism termed early contact inhibition (ECI) (14). cer-resistant African mole rat, Heterocephalus glaber, the naked Naked mole rat cells proliferate slowly in culture and display hy- mole rat in which cells display hypersensitivity to contact inhibi- persensitivity to contact inhibition (14). Abrogation of ECI makes tion. Sequestration of p53 and Rb proteins using SV40 large T naked mole rat cells more prone to malignant transformation. antigen completely rescued necrotic cell death. Our results suggest Naked mole rats inhabit East Africa and belong to the Hys- that cancer resistance of BMR is conferred by massive necrotic re- tricognath group of rodents, which also includes guinea pigs. Na- sponse to overproliferation mediated by p53 and Rb pathways, ked mole rats are evolutionarily distant from BMR, which is closer and triggered by the release of IFN-β. Thus, we have identified to Old World mice and rats. Both mole rats, however, lead sub- a unique mechanism that contributes to cancer resistance of this terranean lifestyles protected from predators and the above- subterranean mammal extremely adapted to life underground. ground extreme climatic fluctuations, which allowed these species to evolve extreme longevity and cancer resistance. aging | necrosis Here we set out to investigate cancer resistance in the BMR. The analysis of BMR-cultured cells reveals that, unlike the naked lind mole rats, Spalax (BMR), belong to a family of sub- mole rat, BMR fibroblasts do not display early contact inhibition, Bterranean rodents of the Muroidea superfamily, prevalent in but instead proliferate rapidly for 7–20 population doublings the Middle East, ranging in the Eastern Mediterranean and (PDs) after which the cells begin to secrete IFN-β and the culture North Africa. BMRs are solitary mammals that spend their lives undergoes concerted cell death (CCD), which is characterized by in underground burrows. Living under extreme hypoxic con- a large fraction of necrotic cells. Inactivation of Rb and p53 ditions, BMRs evolved strong hypoxic tolerance (1, 2). tumor suppressors rescued the cell-death phenotype. Our results BMRs are very long-lived for their size. The maximum lifespan suggest that the BMR has evolved a unique anticancer mecha- documented for the animals kept in our animal facility is 21 y (3). nism mediated by strong induction of the necrotic cell-death In comparison, mice and rats belonging to the same superfamily response to hyperproliferation. Furthermore, we conclude that have a maximum lifespan of 4 y (4, 5). Furthermore, BMRs show the two long-lived cancer-resistant rodents have achieved their a striking resistance to cancer. Our observation of thousands of cancer resistance by two distinct mechanisms. captive animals did not show a single case of spontaneous tumor Results development over a 40-y period. Cancer accounts for ∼23% of human mortality (6). In mice and rats, cancer mortality is very BMR Fibroblasts Do Not Show ECI. To understand the molecular high, reaching 90% in some strains (7, 8). mechanism responsible for the cancer resistance of BMR, we fi Animals have evolved multiple mechanisms to protect them- isolated primary broblasts from BMR lung and skin and ex- selves from cancer. These mechanisms include cell-cycle check- amined their proliferation in culture. The experiments were performed on fibroblasts isolated from two animals of each points, DNA repair, programmed cell death, and replicative fi senescence controlled by a network of tumor-suppressor genes, species: Spalax judaei and Spalax golani (2). All broblast lines showed similar growth characteristics; therefore, we will refer to such as p53 and Rb. Anticancer adaptations differ between spe- fi fi cies, which may explain the differences in cancer susceptibility (9– them as BMR broblasts. BMR broblasts proliferated rapidly 11). Mice have been used extensively as models for cancer research. However, mice are more prone to cancer and are likely to possess fewer anticancer defenses, thus limiting a potential for Author contributions: V.G., A.V.G., E.N., and A.S. designed research; C.H., X.T., J.A., and A.S. performed research; A.V.G. and E.N. contributed new reagents/analytic tools; V.G., discovery of novel anticancer pathways. Hence, there is great value E.N., and A.S. analyzed data; and V.G., E.N., and A.S. wrote the paper. in studying anticancer mechanisms in cancer-resistant species. The authors declare no conflict of interest. Subterranean mammals are good candidates for these studies. 1To whom correspondence may be addressed. E-mail: [email protected], We showed previously that the BMR p53 gene contains an [email protected], or [email protected]. arginine to lysine substitution at a site corresponding to human 2Present address: Department of Genetics and Complex Diseases, Harvard School of Public p53 position 174, where mutations are often found in human Health, Boston, MA 02115.

19392–19396 | PNAS | November 20, 2012 | vol. 109 | no. 47 www.pnas.org/cgi/doi/10.1073/pnas.1217211109 Downloaded by guest on September 29, 2021 with no attached cells remaining within 4 d of the onset of cell A Sparse Confluent death. Our laboratory routinely cultures primary fibroblasts from 20 rodent species, and we have never observed such a synchronous death of cell cultures. We termed this phenom- “ ” Spalax enon concerted cell death (CCD). Our standard growth media judaei for all primary rodent fibroblasts is EMEM supplemented with 15% (vol/vol) FBS. In an attempt to find growth conditions where the cells would not die, we have also cultured BMR cells in low-serum and serum-free fibroblast growth media. However, variations of cell culture media changed the lifespan by a few PDs, after which all cultures invariably underwent CCD (Fig. 1B). Spalax To identify the type of cell death in BMR cultures, we col- golani lected the dying cells, stained them with Annexin-V and propidium iodide (PI), and then analyzed by flow cytometry. The cell death was preferentially necrotic, with 46% of the cells dying by necrosis and 28% by apoptosis (Fig. 2B, control samples).

CCD Is Abrogated by SV40 Large T Antigen. We next tested whether CCD is controlled by the major tumor suppressor pathways, Mus musculus A Large T

Day 2 Day 7 30

B 28 Mouse 24 20 Control 20 BMR 1 Large T K1 16 2 NMR 10 12 4 3 5 8 6 Population Doubling 7 Population Doubling 4 0 0 20 40 60 80 100 120 0 0 20 40 60 80 Time, days Time, days B Fig. 1. BMR fibroblasts display a unique concerted cell death phenotype. 80 All cells were cultured at 3% oxygen concentration. (A) Fibroblasts of both S. judaei and S. golani species become confluent at high cell density and do not undergo early contact inhibition typical of naked mole rat cells. 60 Cells were seeded at 5 × 105 cells per 10-cm dish and photographed at indicated time points. (Magnification: 100×.) (B)BMRfibroblast cultures undergo CCD irrespective of growth conditions. Mouse, denotes mouse 40 fibroblasts, and NMR denotes naked mole rat fibroblasts cultured under our standard conditions of 15% FBS and EMEM media from ATCC. Blue

growth curves correspond to BMR fibroblasts cultured in different con- Cell death, % centrations of FBS and either EMEM media from ATCC or low-serum fi- 20 broblast growth media (FGM) from Lonza. FGM media was used with different concentrations of fibroblast growth factor indicated by (no. in blue) -F or 1/2F (1), 15% FBS, FGM (2), 15% FBS, FGM-F (3), 2% FBS, FGM 0 (4), 15% FBS, EMEM (5), 2% FBS, FGM 1/2F (6), 5% FBS, EMEM (7), 0% FBS, FGM. BMR cells grew rapidly for several PDs, then invariably stopped proliferation and underwent CCD. Control Control Large T Large T Large T K1 Large T K1

and reached high cell density on a plate (Fig. 1A). Cell density MEDICAL SCIENCES fi was similar to the density attained by mouse broblasts. We Day 47 Day 67 concluded that BMR fibroblasts, unlike naked mole rat fibroblasts, do not display the ECI phenotype. Therefore, BMR is Fig. 2. CCD requires Rb and p53 pathways. (A) At day 27 BMR cells were likely to have evolved a distinct anticancer mechanism. stably transfected with plasmids encoding SV40 LT antigen, Large T K1 (a mutant version of LT antigen that inactivates p53 but not Rb), and a control plasmid. Cells transfected with the wild-type LT did not undergo CCD. (B) BMR Cells Undergo Concerted Cell Death. BMR fibroblasts prolif- – CCD occurs by necrosis and apoptosis. Cell death was analyzed by Annexin-V/ erated rapidly for 7 20 PDs, and then the cells arrested proliferation PI staining and flow cytometry in actively growing (day 47) and dying (day for ∼3 d, after which all cells on the plate detached and died, 67) BMR cultures shown in A.

Gorbunova et al. PNAS | November 20, 2012 | vol. 109 | no. 47 | 19393 Downloaded by guest on September 29, 2021 Rb and p53. We transfected BMR cells with SV40 large T an- IFN-β was determined via a calibration curve built by adding hu- tigen (LT) or its mutant derivatives and selected chromosomal man IFN to VSV-GFP–infected HT1080 cells (Fig. 4B). Young, integrants. Wild-type LT is a viral oncoprotein that binds and growing BMR cultures did not release any detectable IFN, but just inactivates both p53 and pRb. The mutant derivative LTK1 before the onset of CCD BMR cells secreted high levels of IFN-β inactivates only p53, and LTΔ434–444 inactivates only pRb and (Fig. 4A). In the second assay, BMR-conditioned media was added its family members (p107 and p130) (15). BMR cells expressing to HEK-blue cells containing a β-galactosidase gene under an LT proliferated continuously for over 50 PDs and did not un- IFN-β–responsive promoter. Young, growing BMR cultures did dergo CCD (Fig. 2). LTK1 did not rescue CCD, but rather ac- not induce β-galactosidase above the control level, but conditioned celerated its onset (Fig. 2), but LTΔ434–444 was toxic to the cells media from the dying cultures showed a threefold elevated ex- and did not yield any chromosomally integrated clones despite pression of the reporter gene. Notably, transfection with SV40 LT β multiple attempts. This result indicates that both Rb and p53 antigen that rescues CCD also abrogated IFN- release (Fig. 4C). pathways must be inactivated to abrogate CCD. Finally, we tested whether BMR-conditioned media from dying cells would induce CCD in young BMR cells or in cells for CCD Is Not Caused by Telomere Shortening. We then tested whether other species. Fresh media, media conditioned by young, grow- CCD is induced by rapid telomere shortening. Importantly, ing BMR cells, and media conditioned by dying BMR cells were BMR cultures expressed high levels of telomerase activity (Fig. added to young BMR, mouse, or human cells. Media condi- 3A), as expected for a small-sized rodent (10). In addition, time- tioned by dying BMR cells caused massive necrotic cell death of resolved ﬂuorescence (TRF) assay showed that BMR has telo- young BMR cells, and massive apoptotic death of mouse cells meres with a mean length of 50 kb, which is much longer than (Fig. 4D). Media conditioned by dying BMR cells did not cause telomeres in the species with replicative senescence, such as massive death of human cells, which is likely explained by the divergence between rodent and human interferons. In summary, humans (Fig. 3B). Furthermore, telomere length did not change these results indicate that CCD is triggered by IFN-β release. in cultured cells undergoing CCD. These results indicate that CCD is not triggered by telomere shortening. Discussion

Concerted Cell Death of BMR Cells Is Mediated by Release of IFN-β. In this study we show that cells of a cancer-resistant rodent, BMR, display an unusual cell death mechanism that rapidly We then set out to identify the mechanism of CCD. We hy- wipes out entire cultures of cells, leaving no survivors. CCD pothesized that the pattern of cells dying synchronously is con- occurs by necrosis and is triggered by the release of INF-β. This sistent with a response to IFN. We tested whether BMR cell β fi response required functional p53 and Rb pathways. When cells cultures secrete IFN- using two assays. In the rst assay, HT1080 are cultured in vitro they are subjected to growth factors from cells were infected with vesicular stomatitis virus encoding a GFP FBS and are forced to proliferate because of frequent subculture gene (VSV-GFP). The number of cells containing green fluores- fi fl at low cell density. These strong progrowth signals may be rec- cent virus particles was quanti ed by ow cytometry. HT1080 cells ognized by BMR cells as a potentially oncogenic signaling im- were incubated overnight with the media conditioned by BMR balance and trigger a cell death response. This mechanism cells at different stages of growth. HT1080 cells were then infected resembles oncogene-induced senescence of human cells, where fi with VSV-GFP, and the presence of IFN was quanti ed by the cells enter irreversible arrest in response to hyperproliferative ability of the conditioned media to halt VSV-GFP infection and + signals, such as expression of activated oncogenes (16, 17). An- reduce the number of GFP cells (Fig. 4A). The number of units of other similarity with oncogene-induced senescence is that CCD of BMR cells is independent of telomere shortening and occurs in cells with long telomeres. We hypothesize that in vivo CCD fi A B ef ciently clears away premalignant cells contributing to the BMR BMR Human cancer resistance of the BMR. Cell culture experiments revealed that a similar necrotic cell death takes place in primary BMR cell G D G D Y S cultures upon extensive in vitro proliferation. HeLa kb Strikingly, BMR cells use necrosis rather than apoptosis to oncogenic insults. This finding can be explained by the unusual se- 150 quence of BMR p53, which is deficient in activating an apoptosis 100 cascade, and evolved as an adaptation to subterranean life under hypoxic conditions (12). Nevertheless, necrotic death of BMR 50 cells requires a functional p53 pathway. Despite necrosis being 33 commonly viewed as less precise or as an inefficient way of elim- 25 inating unwanted cells, BMRs have evolved a highly efficient an- 19 15 titumor mechanism based on necrotic response. An advantage of 12 necrosis could be in eliminating all cells surrounding the pre- 10 malignant lesion, which may provide an added antitumor effect 8 7 by eliminating reactive tumor stroma, including tumor-activated 6 fibroblasts (18). 5 In the future, it would be interesting to move these studies into in vivo system by testing whether CCD protects BMRs from 4 chemically induced carcinogenesis. It would also be interesting to mix BMR cells with malignantly transformed mouse cells and test whether BMR cells suppress carcinogenesis in a xenograft Fig. 3. CCD of BMR cells is not caused by telomere shortening. (A) BMR mouse model. fibroblasts have endogenous telomerase activity. Telomeric repeat amplification protocol assay was performed on extracts from growing (G) or dying Remarkably, cells of another cancer-resistant subterranean (D) BMR cells, and HeLa cells as a positive control. (B) Telomeres do not rodent, the naked mole rat, do not display CCD, and achieve shorten in dying BMR cells. Telomere length in growing (G) and dying (D) high PDs in culture (11). Furthermore, naked mole rat cells BMR cells was measured using TRF assay. Young (Y) and senescent (S) human easily undergo apoptosis and do not favor necrosis in response to fibroblasts were used as a reference. stress (14). This finding is consistent with the naked mole rat p53

19394 | www.pnas.org/cgi/doi/10.1073/pnas.1217211109 Gorbunova et al. Downloaded by guest on September 29, 2021 A B 20 60

160 50 16 40 120 12

, Units/ml 30

80 % GFP, 8 20 10 4 40 Population Doubling Interferon- 0 0 40 20 60 80 120 140 160 180 0 0 100 0 10 20 30 40 50 60 70 80 Interfiron- , Units/ml Time, days C D 0.8 80 0.6 60

0.4 40 -gal activity, OD -gal activity, Cell death, % 0.2 20

0 0 HEK-blue Conditioned Dying Dying media: Dying hINT- Control Control Control Control BMR LT BMR LT Growing Growing Growing

BMR dying BMR Mouse Human BMR growing E Control Dying

BMR

Mouse

Fig. 4. CCD is triggered by release of IFN-β by BMR cells. (A) IFN release coincides with the onset of CCD. The growth curve for BMR cells is overlaid on the bar graph showing IFN levels in the media. IFN levels were measured by VSV-GFP assay. In this assay HT1080 cells are incubated with fresh media, or conditioned media containing INT-β; the cells are then infected with a GFP-encoding VSV, and the level of IFN in the test media corresponds to the reduction in the number of GFP positive HT1080 cells. (B) Calibration curve used to quantify the amount of BMR IFN in A. The curve was built by performing a VSV assay with known amounts of human INT-β.(C) IFN release by dying BMR cells determined by HEK-blue assay, which measured the induction of β-gal reporter in HEK cells under IFN-inducible promoter. Control indicates untreated HEK-blue cells. hINT-β are HEK-blue cells to which 300 U/mL of human IFN-β was added. Other bars show HEK-blue cells treated with BMR-conditioned media. (D) Conditioned media from dying BMR cells causes death of mouse cells. Media conditioned by BMR cells was added to growing BMR, mouse, or human fibroblasts. Cell death was quantified by Annexin-V/PI staining and flow cytometry. (E) Microscopic examination of CCD. Photographs of early passage mouse and BMR cells before and after addition of conditioned media from dying BMR cells. (Magnifi- cation: 100×.) Upon addition of the BMR conditioned media mouse cells display typical apoptosis with membrane blebbing, but BMR cells show pre- dominantly necrotic cell death with cells detouching from the plate and breaking up without membrane bebbing.

gene, which has arginine in position 174 (19), and is thus similar superfamilies have shown that these mechanisms are distinct for to the mouse or human p53 in its propensity to induce apoptosis. each species, as they evolved phylogenetically independently in Naked mole rat cells display early contact inhibition that acts as each species, possibly associated with hypoxic conditions un-

an additional tumor suppressor mechanism in this species. In derground and the species longevity. We speculate that many MEDICAL SCIENCES contrast BMR cells proliferate to high cell density. Thus, BMRs unique anticancer adaptations will be found in long-lived rodent and naked mole rats have evolved two distinct anticancer species. This knowledge could then be used for treatment or mechanisms that provide remarkable cancer resistance to these prevention of cancer in humans. small rodents. Our earlier comparative studies of 15 rodent species suggested Experimental Procedures that small, long-lived rodents evolve novel anticancer mecha- Cell Isolation and Culture. Primary blind mole rat (BMR) and mouse ﬁbroblasts nisms (10, 11). The current in-depth analysis of two small, long- were isolated from lung and under arm skin, as described previously (20). The lived species of subterranean rodents belonging to different experiments were performed on ﬁbroblasts isolated from two animals of

Gorbunova et al. PNAS | November 20, 2012 | vol. 109 | no. 47 | 19395 Downloaded by guest on September 29, 2021 each BMR species: Spalax judaei and Spalax golani (2) and three mice Mus IFN-β Assays. Vesicular stomatitis virus encoding a GFP gene assay. Vesicular musculus. All BMR fibroblast isolates showed similar growth characteristics. stomatitis virus encoding a GFP gene (VSV-GFP) was previously described (21). Normal human fibroblasts were HCA2 neonatal foreskin fibroblasts. It consists of VSV with GFP linked to the cytoplasmic domain of the VSV-G Human Fibrosarcoma (HT-1080) cells were purchased from American Type protein. The addition of GFP to VSV-G does not cause a reduction in titer, is Culture Collection (ATCC CCL-121). stable through multiple passages, and is incorporated into virions with al- Under standard conditions, all primary fibroblasts were cultured at 37 °C, most the same efficiency as wild-type VSV-G. The virus was expanded as μ × 6 5% CO2,3%O2, on treated polystyrene culture dishes (Corning) in EMEM follows: 200 L of the virus was added onto 1.5 10 HeLa cells plated media (ATCC) supplemented with 15% FBS (Gibco), nonessential amino 2 d earlier; 48 d after infection, media with the virus was collected, filtered, acids, sodium pyruvate, 100 units/mL penicillin, and 100 μg/mL streptomycin and stored at −80 °C. The titer of the virus was determined for individual (Gibco) or FGM-2 BulletKit (Lonza) supplemented with 15% FBS (Gibco). cell lines using standard plaque assay. Aliquots of VSV stock were stored at − For experiments with different types of media (Fig. 1B), BMR cells were 80 °C until use. cultured in different concentrations of FBS (0%, 2%, or 15%) and either For the experiments, the viruses were thawed slowly on ice and diluted μ EMEM media from ATCC or low-serum fibroblast growth media (FGM) from in EMEM medium to obtain a working stock of 100 pfu/ L. VSV was used Lonza. FGM media was used with different concentrations of fibroblast according to bio-safety procedures in a P2-level safety facility/room. The use growth factor. In addition, we unsuccessfully tried to grow BMR cells in low of VSV-GFP in this project was reviewed and approved by the Institutional serum growth medium 106 (Gibco) supplemented with different concen- Biosafety Committee at the University of Rochester. trations of FBS (0%, 2%, or 15%) and with different concentrations of low For the assays, media conditioned with BMRs at different stages of growth, fi – serum growth supplements (Gibco). mouse, or human HCA2 broblasts were collected after 3 5 d of growing + We did not observe significant differences in the growth of BMR cells on cells, spinned down to remove debris, and stored at 4 °C. For the VSV-GFP different surfaces, including treated polystyrene culture dishes (Corning), BD infection we used HT1080 cells, that were seeded at a density of 60,000 cells PureCoat Amine (positively charged surface), BD PureCoat Carboxyl (nega- per well in six-well plates in EMEM with 15% FBS and incubated for 24 h. tively charged surface), collagen, fibronectin, gelatin, and glass. Then, the media was discarded and the conditioned media was added to each well. In addition, we used human IFN-β human (100 U per well) as a positive control and human IFN-γ (100 U per well) as a negative control. Analysis of Cell Growth. Cells were seeded at 5 × 105 cells per 100-mm dish. After 3–4 h of incubation in conditioned media, cells were infected with When cells reached 80% confluence, they were harvested, counted, and the VSV-GFP at a multiplicity of infection 0.1 or 1 for a 12–16 h period. GFP signal number of population doublings was calculated. appears in infected cells after 12–16 h, and the cells die about 6 h later. FACS analysis was performed 16 h after infection to quantify GFP+ cells. Analysis of Telomeres. Telomeric repeat amplification protocol and time-re- HEK-Blue cells assay. HEK-Blue IFN-α/β cells (Invivogen) allow the detection of solved fluorescence assays were performed as previously described (11). bioactive human type I IFNs by monitoring the activation of the ISGF3 pathway. The assay was performed according to the manufactured protocol Analysis of Cell Death. Cell death was analyzed using Annexin-V-FLUOS assay (Invivogen). Briefly, HEK-Blue cells were plated in 96-well plates and in- ’ fl fl kit (Roche) according to the manufacturer s instructions. Brie y, oating and cubated overnight. Then, 20 μL of test medium was added to each well. adherent cells were harvested, stained with Annexin-V and propidium io- Human IFN-β was used as a positive control and human IFN-γ was used as fl dide, and analyzed on a BD Biosciences FACS Canto ow cytometer. a negative control. Cells were incubated overnight. The next day, 30 μLof the supernatant from Hek-Blue cells was added to 170 μL of Quanti-Blue × 5 Transfections. Fibroblasts were seeded at 5 10 cells/100-mm plate 2 d be- Reagent (Invivogen) for 3 h at 37 °C. The colorimetric reaction was measured 6 fore transfection. For transfection, cells were harvested, counted, and 10 at 650 nm on a plate reader. cells were transfected with 5 μg of plasmid DNA using Amaxa Nucleofector II on program U-020 and solution NHDF (Amaxa). Transfections included ACKNOWLEDGMENTS. This work was supported by grants from the National a mock with no DNA, pSG5 Large T (Addgene 9053), pSG5 Large T K1 Institutes of Health and Ellison Medical Foundation (to V.G.); an Ellison (Addgene 9055), pSG5 Large TΔ434–444 (Addgene 9054), and no electro- Medical Foundation grant (to A.S.); and the Ancell-Teicher Research Foun- poration control. dation of Genetics and Molecular Evolution (E.N.).

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