The proapoptotic function of SAP providesa clue to the clinical picture of X-linkedlymphoproliferative diseaseNoemi Nagya, Liudmila Matskovaa, Lorand L. Kisa, Ulf Hellmanb, George Kleina,1, and Eva Kleina

aDepartment of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institute, SE-171 77 Stockholm, Sweden; and bLudwig Institute for Cancer Research,Uppsala University, SE-751 24 Uppsala, Sweden

Contributed by George Klein, May 28, 2009 (sent for review March 31, 2009)

Deletion or mutation of the SAP gene is associated with the X-linkedlymphoproliferative disease (XLP) that is characterized by extremesensitivity to Epstein-Barr virus (EBV). Primary infection of the af-fected individuals leads to serious, sometimes fatal infectious mono-nucleosis (IM) and proneness to lymphoma. Our present resultsrevealed a proapoptotic function of SAP by which it contributes to themaintenance of T-cell homeostasis and to the elimination of poten-tially dangerous DNA-damaged cells. Therefore, the loss of thisfunction could be responsible for the uncontrolled T-cell proliferationin fatal IM and for the generation of lymphomas. We show now therole of SAP in apoptosis in T and B lymphocyte-derived lines. Amongthe clones of T-ALL line, the ones with higher SAP levels succumbedmore promptly to activation induced cell death (AICD). Importantly,introduction of SAP expression into lymphoblastoid cell lines (LCL)established from XLP patients led to elevated apoptotic response toDNA damage. Similar results were obtained in the osteosarcoma line,Saos-2. We have shown that the anti-apoptotic protein VCP (valosin-containing protein) binds to SAP, suggesting that it could be instru-mental in the enhanced apoptotic response modulated by SAP.

activation induced cell death � apoptosis � lymphomas � p53

X -linked lymphoproliferative disease (XLP) is a rare primaryimmunodeficiency that manifests most often after primary

Epstein-Barr virus (EBV) infection (1). The XLP phenotypeincludes fatal infectious mononucleosis (fatal IM) (50%), malignantlymphomas (30%), and dys-gammaglobulinemia (30%). Mutationsfound in XLP patients in the gene located on Xq25 identified themolecular basis of the disease (2–4). The XLP gene (SH2D1A)codes for a short protein, SAP, of 128 aa, that contains an SH2domain through which it binds to receptors of the signaling lym-phocytic activation molecule (SLAM and CD150) family, including2B4 (CD244), NTB-A, CD84, Ly9 (CD229), and CD2-like recep-tor-activating cytotoxic cells (CRACC). These molecules are ex-pressed in cells of the immune system [for a review see Veillette (5)].

The majority of point mutations in the SAP gene lower markedlythe stability of the protein, leading to its loss (6). Other mutationsdisrupt its binding to the SLAM family receptors.

IM is an immunopathological disease, characterized by lympho-cytosis as a result of expansions of NK cells and CD8� T lympho-cytes in response to the primary EBV infection (7). The severity ofthe disease varies, but it subsides regularly. In contrast, the courseof primary EBV infection in XLP patients is always severe. In 50%of the cases, it leads to death (fatal IM) caused by overwhelmingcytotoxic T-cell activation and multiorgan failure.

In addition to the intense proliferation of T cells, the highfrequency of apoptosis prone T lymphocytes is an important featureof IM (8). Upon resolution of the disease, the absolute number ofcirculating T cells and the relative proportion of CD4� and CD8�returns to normal (9).

The NK cells of XLP patients are functionally impaired (10), andit has been shown that SAP controls the cytotoxic functions of NKand T cells (11, 12). Thus, lack of the SAP protein explains the de-creased cell mediated cytotoxic activity in XLP patients. Since T

and NK cells are held responsible for the elimination of EBV-infected B cells, it was suggested that mainly the inefficiency of theseeffector cells is responsible for the prolonged, severe and often fataldisease course.

Assuming that SAP has further functions, in that it may controlthe amplitude and duration of the immune response during theprimary EBV infection, we tested whether the increase of SAPexpression in the late phases of T-cell activation, correlates with theresolution of the cellular immune response.

Our earlier work showed that wt p53 induces SAP in lymphoidcells (13), suggesting the possibility that SAP is an executormolecule in some of the several p53 controlled pathways. P53 is amultifunctional regulator having a central role in several cellularpathways such as cell-cycle control, apoptosis, and DNA repair,contributing critically to the maintenance of genomic stability andsuppression of cell transformation.

We studied therefore the effect of SAP overexpression in differ-ent cell types in situations that could reflect the course of 2 eventsin XLP patients: (i) the homeostasis of T cells in IM and (ii) the fateof cells subjected to genotoxic stress in the process of lymphomadevelopment.

Our results identified SAP as a proapoptotic cellular protein,suggesting an unexpected role in the pathology of XLP disease.

ResultsSAP Is Up-Regulated in PHA-Activated T Cells. Cognate stimulation ofT cells induces their activation and proliferation. Ultimatelyhowever, activated T cells succumb by apoptosis and the T-cellnumber declines. Our earlier finding that SAP is increased in thelate phases of T-cell activation (14) suggested that in addition toits known function in regulating signals at the initiation of T-cellactivation, SAP may regulate T-cell homeostasis. This assump-tion is supported by the kinetic analysis of PHA-activated T cellsin that the expression of SAP, and the proliferation of the cells(measured by 3H-Thy incorporation) showed a negative corre-lation (Fig. 1A).

P53 is up-regulated in the activated T cells (Fig. 1B), andaccording to our earlier finding, p53 induces SAP expression.Therefore, it is likely that SAP up-regulation in activated T cellsmay involve p53.

Increased Expression of SAP Does Not Induce Changes in SurfaceMarker Expression of CCRF-HSB2 Tumor T-cell Line. To study moredirectly the proliferative/ apoptotic role of SAP in T cells, weestablished T-cell lines with different SAP levels. The T-ALL tumorcell line CCRF-HSB2 expresses relatively low level of SAP. In 2independent experiments, we introduced SAP by retrovirus deliv-

Author contributions: N.N., L.M., G.K., and E.K. designed research; N.N., L.M., L.L.K., andU.H. performed research; N.N., U.H., G.K., and E.K. analyzed data; and N.N., G.K., and E.K.wrote the paper.

The authors declare no conflict of interest.

1To whom correspondence should be addressed. E-mail: georg.klein@ki.se.

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ery to the cells and obtained 4 sublines with low SAP (CCRF-HSB2-vector) and 4 sublines with high SAP (CCRF-HSB2-SAP)expression. In subsequent experiments, we used 2 low and 2 highSAP expressor sublines (Fig. 2).

The phenotype of the 4 sublines determined by FACS showed nodifferences in expression of CD25, CD69, CD95, ICAM-1, LFA-1,SLAM, and 2B4.

The expression of the anti-apoptotic proteins Bcl-2 and Bcl-xl,detected by immunoblot, did not differ either in the 4 lines (Fig. 2).

CCRF-HSB2 Clones Expressing High SAP Levels Are More Sensitive toActivation-induced Cell Death. T-cell homeostasis is controlled byactivation induced cell death (AICD), in which apoptotic process isinitiated when activated T cells are restimulated. We used the in vitromodel of AICD that is activation of cells by PMA� ionomycin or PHA.

During the 6-day period of the test, the number of viable cells inthe culture initiated with the high SAP expressor hardly increased(Fig. 3A). This was due, at least in part, to higher death rate in thepopulation of the high SAP expressors (85%) compared with lowerSAP expressors (35%) following activation with 100 ng/mL PMA� 4 �M ionomycin. The results with PHA activated (5 �g/mL) cellswere similar (40% versus 18%).

By definition, the AICD occurs through apoptosis. Therefore, inthe next experiment, we determined the kinetics of the appearanceof Annexin V-positive cells in PMA � ionomycin-treated cultures.The kinetics and dose dependence of appearance of AnnexinV-positive cells showed that the highest concentration of drugs (200ng/mL PMA � 8 �M ionomycin) induced massive apoptosisalready after 2.5 h (Fig. 3B). In the cultures treated with the lowerdoses, apoptotic cells appeared after 6 and 22 h. At all time pointsand concentrations, significantly higher percentages of apoptoticcells were detected in the cultures of the cells that expressed higherSAP levels (Fig. 3B). This was accompanied by a higher level ofactivation of caspases (Fig. 3C) and PARP cleavage (Fig. 3D). Thus,the levels of SAP in the T cells correlated with sensitivity to AICD.

AICD involves the function of the death receptor CD95. There-fore, we tested the sensitivity of the CCRF-HSB2 clones to theagonistic anti-CD95 Ab (anti-APO-1). This comparison was feasi-ble because CD95 expression was similar on the sublines. Ligationof CD95 induced higher number of apoptotic cells in the clones withhigher SAP level (22% versus 12%), further substantiating theinvolvement of SAP in AICD.

SAP Controls Cell Cycle/Apoptosis in DNA-damaged Saos-2 Cells. Wereportedearlier thatSAPisap53target in lymphoidcells.Thus,SAPcould execute some of the p53 functions. We tested this assumptionin the osteosarcoma cell line, Saos-2, which lacks p53. We inducedDNA damage by irradiation in Saos-2 cells into which SAP wasintroduced by retroviral transduction and could therefore investi-gate the direct involvement of SAP without the contribution of p53.

In short-term experiments, the division of cells was monitored bythe dilution of the membrane dye PKH26. The SAP positive cellsdid not divide during 2 days following DNA damage (400 rad)whereas the Saos-2-vector cells divided as indicated by the decreaseof fluorescence intensity (Fig. 4A, Lower).

The cells were also followed for a longer time period. Seven daysafter irradiation, the Saos-2-vector cultures contained several grow-ing colonies, whereas no colonies were seen in the Saos-2-SAPcultures (Fig. 4B, Left). Ten days after the initiation of cultures,viable colonies were detected by MTT assay only in the SAP-negative cultures (Fig. 4B, Right).

We also followed the fate of the irradiated Saos-2 cells during anextended period. Three days after irradiation of the cells, cultureswere initiated with equal cell numbers and kept for 28 days. Thetotal number of cells was lower in the Saos-2-SAP cultures com-pared with Saos-2-vector cells (Fig. 4C), indicating that the expres-sion of SAP was required to compromise cell proliferation afterirradiation.

The experiments with the Saos-2 lines suggest thus that SAP isinvolved in the cell cycle control/apoptosis of DNA-damaged cells.

LCLs Derived from XLP Patients Are Not Arrested in the G2/M Phaseof the Cell Cycle Following DNA Damage Induced by �-irradiation. Toelucidate the functional consequences of a mutated SAP, wecompared the cell cycle distribution of EBV-transformed lympho-blastoid cell lines (LCLs) derived from healthy donors and fromXLP patients (carrying wt p53), following DNA damage (double-strand breaks) induced by �-irradiation. We have previously shownthat LCLs do not express SAP, but it can be induced when cells areexposed to DNA damage because p53 activates the SAP promoter(13). Since the SAP gene is mutated or it is deleted in the XLPpatients, they will lack or express mutated/truncated SAP followingDNA damage. Although the healthy donor derived LCLs arrestedin the G2/M phase of the cell cycle following irradiation (Fig. 5A),this did not occur with the XLP patient-derived LCLs (Fig. 5B).This result suggested the involvement of SAP in the cell cyclecontrol/DNA repair/apoptosis in that a nonfunctional SAP wouldbe unable to induce a normal DNA damage response, manifestedby failure to induce growth arrest in G2/M phase of the cell cycle.

Fig. 1. Up-regulation of SAP and cessation of proliferation occur in parallel inactivated normal T cells. (A) T cells were isolated from peripheral blood of healthydonors and cultured in medium without or with 1 �g/mL PHA for the indicatedtime periods. SAP and �-actin were detected in inmmunoblots. The bands werequantified with densitometer and the SAP/actin ratio was calculated. 3H-Thymidine incorporation of 105 cells was determined on the indicated days. Errorbars represent SD of cpm values for 5 replicate cultures. Representative result ofat least 7 experiments. (B) p53 expression is up-regulated in activated T cells.Peripheral blood derived T cells were cultured in medium alone or were activatedwith 1 �g/mL PHA for 5 days. Samples were collected and lysates correspondingto 2 � 105 cells were tested in immunoblot for SAP and p53 expression. Repre-sentative blot of 3 experiments.

Fig. 2. SAP, Bcl-2 and Bcl-xL expression in retrovirus transduced CCRF-HSB2clones.Lysatespreparedfromcells transducedwithcontrol retrovirus (CCRF-HSB2vect.) or with retrovirus carrying the SAP gene (CCRF-HSB2-SAP) were tested forSAP, Bcl-2, and Bcl-xL expression by immunoblot. Actin was used as a control forequal loading. The numbers in the name identify the different clones.

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Constitutive Expression of SAP Renders LCLs More Sensitive to Apo-ptosis. To confirm that SAP is responsible for cell cycle regulation/apoptosis in lymphoid cells, we introduced SAP into LCLs estab-lished from XLP patients (TR and K001-BK) with the help of retro-virus delivery system. The cells constitutively expressed a low, butstable level of SAP. Twenty-four hours after irradiation of these cellswith 1,000 rad, we tested the cell cycle distribution. The LCLs thatexpressSAPshowedasignificantlyhighernumberofdeadcells (withDNA content in the sub G1 fraction) (Fig. 6A). Similar results wereobtained with the other LCL, K001-BK, where the SAP expressorcultures had higher percentage of dead cells at all time points testedafter irradiation (Fig. 6B). These results strongly suggest that SAPis involved in the elimination of DNA damaged cells.

SAP Binds VCP, a Protein with Anti-Apoptotic Function. In an attemptto identify the apoptosis regulatory pathway(s) with which SAP

might interfere, we looked for SAP-binding partners by coimmu-noprecipitation. CCRF-HSB2 cells were lysed and precipitatedwith anti-SAP antibody and with normal rabbit serum (NRS) as anegative control. After SDS-PAGE, the proteins were visualized bysilver staining, and bands present in the anti-SAP precipitates butabsent from NRS precipitates were subjected to mass spectrometryanalysis. As a validation of the method’s specificity, a protein bandof �15 kDa was confirmed as SAP. One of the coprecipitatedproteins, with a molecular weight of �100 kDa, was identified asvalosin-containing protein (VCP). This protein has been shown tobe involved in regulation of apoptosis, making it an attractivecandidate by which SAP could regulate apoptosis. Subsequently, weprecipitated endogenous SAP from CCRF-HSB2 cells and tran-siently transfected SAP from Saos-2 cells. The immunoprecipitatedsamples were probed in western blot with anti-VCP antibodies andthis confirmed the binding of SAP to VCP (Fig. 7A). However, in

Fig. 3. CCRF-HSB2 clones with ‘‘high’’ SAP levels are more prompt to apoptosis following activation. (A) The number of living cells in activated cultures ofCCRF-HSB2-SAP-1 (expressing ‘‘high’’ SAP) increased more slowly. CCRF-HSB2 clones (vector-1 and SAP-1) were activated with 100 ng/mL PMA � 4 �M ionomycin. Thecell number was determined at the indicated time points. (B) CCRF-HSB2-vector and -SAP lines were activated with the indicated concentrations of PMA � ionomycinfor 2.5 h (Left), with 100 ng/mL PMA � 4 �M ionomycin for the indicated time points (Right). Apoptotic cell death was determined by annexin V-FITC staining anddetected by flow cytometry. (C) CCRF-HSB2-vector and -SAP lines were activated with 200 ng/mL PMA � 8 �M ionomycin for 3 h. Caspase activation was determinedwith Guava-MultiCaspase kit following the manufacturers’ instructions. (D) CCRF-HSB2-vector and -SAP lines were activated with 200 ng/mL PMA � 8 �M ionomycinfor 3 h. Lysates corresponding to 2 � 105 cells were tested for PARP expression by immunoblot. The appearance of the shorter, cleaved PARP indicates activation ofexecutor caspases. Figures correspond to 1 representative result of 3 experiments.

Fig. 4. Irradiation induces growth arrest in SAP expressing Saos-2 cells. (A) Saos-2-vector and -SAP cells were labeled with PKH26 membrane dye and irradiated with400 rad. Cells were run in FACS (Upper) or plated for further culture. After 2 days, cells were collected and membrane fluorescence intensities were determined againin FACS (Lower). On the histograms thin line corresponds to the vector- and thick line to SAP expressing Saos-2. (B) No colonies were detected in cultures of irradiatedSAP expressing Saos-2. Cells were irradiated with 400 rad and plated on 12-well plates. Seven days after irradiation cultures were examined/photographed by phasecontrast microscopy. Ten days after irradiation, the same cultures were incubated with 2 �g/mL MTT for 5 h. Living colonies became blue and after discarding themedium cultures were photographed. (C) SAP expressing Saos-2 cells do not survive/proliferate following DNA damage induced by irradiation. Cells (0.75 � 106) weregrown in flasks for 24 h prior irradiation with 400 rad. Three days after irradiation, cells were trypsinized, and cell numbers were equalized for 0.75 � 106. Medium waschanged every 4 days. On day 28 after irradiation, the cell number/ culture was determined. One representative result of 3 experiments.

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the reciprocal experiments, SAP was not detected in the precipi-tates obtained with VCP-specific antibodies. To rule out that thenegative result was because of the specificity and/or affinity of theVCP antibody, we constructed a Myc-tagged VCP plasmid andexpressed it in Saos-2 cells together with SAP. Again, binding ofSAP to Myc-VCP was proven (Fig. 7B), but SAP was not found inthe immunoprecipitates obtained with the anti-Myc reagents. Thismay be because interaction of VCP with SAP is dynamic. Inaddition, endogenous VCP may compete with the Myc-tagged VCPfor binding SAP.

DiscussionWith rare exceptions, all humans carry Epstein-Barr virus (EBV).Primary infection of adolescents and young adults induces a self-limiting disease, infectious mononucleosis (IM) in approximatelyhalf of the individuals (15). The clinical picture of IM is highlyvariable, ranging from mild to severe symptoms (16). Silent infec-tion, that is the rule in young children, can be detected by thedevelopment of humoral and cellular EBV-specific immunity.

XLP has been considered as an EBV-specific immunodeficiency,because the severe, often fatal disease follows EBV infection,leading to the death of the patient in 50% of cases. However,individuals with this genetic defect do not have altered response toother herpesviruses. However, they exhibit humoral immune dys-functions and a 200 -times higher risk for the development ofmalignant lymphomas than the general population.

The symptoms of IM reflect the involvement of the immunesystem. EBV infection elicits a dramatic increase in the numbers ofNK and CD8� T cells. The activated CD8 lymphocytes, which canaccount for �50% of the total blood lymphocyte population (7),

release cytokines that are predominantly of a Th1 type (IFN-� andIL-2) (17, 18) and are thought to induce the clinical features.

The high apoptotic rate of T cells (19, 20), as a consequence ofa markedly decreased expression of Bcl-2 and high levels of FasL, isan important factor in the recovery from IM. Normally, the CD8 T-cell responses subside and in the contraction phase the majority ofvirus-specific CD8 T cells die while a pool of memory cells emerges.It is possible that the regulation of the T-cell dynamic is alsoimpaired in XLP patients. This has already been suggested by theelevated numbers of T cells found in SAP-deficient mice infectedwith either LCMV or with murine gammaherpesvirus-68 (21, 22).

The aim of our studies was to elucidate the role of SAP in thetermination of the intense immune response elicited by primaryEBV infection. We have previously shown that SAP was up-regulated in T cells activated with PHA or with allogeneic irradi-ated LCLs (23). The indication that SAP may be involved in thetermination of T-cell responses emerged from the kinetic studies onSAP levels and cell proliferation, which showed an inverse corre-lation between these 2 parameters. These results suggested that thehigh SAP levels in the late phases of activation have a negativeeffect on cell proliferation/survival.

T-cell homeostasis involves activation-induced cell death(AICD), in which restimulation of the activated T cells initiatesapoptosis. Our comparative experiments with T-cell tumor clonesshowed that T cells with high SAP levels responded more readily inthe AICD test. These results with the tumor lines suggest theinvolvement of SAP in AICD that may be valid for primary T cellsas well. Therefore, the absence of functional SAP in the XLPpatients could allow extended survival of over-activated T cells inIM, and their persistence could lead to the massive tissue infiltratesand organ failures seen in fatal IM.

Fig. 5. LCLs derived from XLP patients do not arrest at the G2/M phase of cell cycle following DNA damage. (A) Healthy donor (LS and Nadia)- and (B) XLP-derivedLCLs (TR and K001-BK) were irradiated with 1,000 rad. Twenty-four hours later, the cell cycle distribution of the samples was analyzed by propidium iodide staining.Histograms show 1 representative result of 3.

Fig. 6. Constitutive expression of SAP in XLP derived LCLs imposes increased apoptosis following DNA damage. Vector retrovirus and SAP expressing retroviruscarrying LCLs (TR and K001-BK) were irradiated with 1,000 rad. After 20 h (A: TR), or 24 h and 48 h (B: K001-BK), cell cycle distribution was analyzed by propidium iodidestaining. Cells with the subG1 DNA content were considered as apoptotic/dead. One representative result of 3 experiments.

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A link between high in vivo SAP levels and rapid apoptosis oflymphocytes in acute IM can be inferred from a recently publishedstudy. Analysis of IM blood revealed that at the time of diagnosis,SAP expression in PBMC was higher than in control subjects. Thiswas probably followed by massive apoptosis in the IM blood, sincea few days after diagnosis (3–16 days), the lymphocyte countsreturned to normal (9).

However, why would the defective AICD, caused by nonfunc-tional SAP, manifest specifically in primary EBV infection? Wepropose that it is because of the exceptional relation of the virus andthe immune system. The virus infects the B cells that becomeactivated and proliferate. The B blasts, present in high numbers,express high levels of costimulatory molecules and therefore stim-ulate the T cells more intensely than in other infections. Thus,following the elimination of the infected B cells, an efficienthomeostasis of the activated T cells is particularly important forrecovery from IM. The evidence presented here suggests a defi-cient-T-cell homeostasis in the individuals that lack SAP. Theextraordinary high numbers of activated T cells will escape AICD,they will survive, leading to the development of fatal IM. Inaddition, the defective cytotoxic function of CTLs and NK cellswould contribute to the vicious cycle, the persistence of EBV-infected B cells would continuously activate T cells.

Similarly, SAP-deficient, OT-I TCR transgenic mouse CD8 Tcells showed a defective AICD, associated with lower levels of p73that influences the mitochondrial cell death pathway (24). Inter-estingly, this phenotype was seen only in primary CD8 T-cellresponses.

It has been assumed that malignant lymphomas appear with veryhigh frequency in XLP patients because of deficient NK and CD8�cytotoxic T cell-mediated control of EBV-infected, proliferating Bcells. However, data are missing to support this hypothesis. Thepresence of EBV in the XLP lymphomas often has not beendocumented and more recent data clearly proves the existence oflymphomas in EBV-negative patients (25). Moreover, in a retro-spective study on XLP patients from the XLP registry, Sumegi etal. found that if FIM is excluded (which occurs only in EBV-positivepatients), the frequency of lymphoproliferative disease is the samein EBV-seronegative and -positive XLP patients (26). Therefore, inaddition to the disturbance in the EBV-specific immune response,the absence of functional SAP has to affect the B cells as well.

In our previous work, we showed that SAP is a target of the tumorsuppressor p53 (13). This finding motivated further experiments to

establish whether SAP is involved in some of the many functions ofp53.

We studied therefore the fate of cells exposed to genotoxic stressin relation to their SAP expression. We previously showed thatLCLs do not express SAP, but as a consequence of p53 activation, itis induced after DNA damage (13). Cell cycle distribution revealedthat in LCLs derived from healthy donors, but not in the LCLsestablished from XLP patients, DNA damage induces G2/M arrest.Even though the same results were seen consistently on 2 healthydonor and 2 XLP derived LCLs, we could not rule out the possi-bility that independently of SAP expression the cell lines differ inother properties. However, results with the Saos-2 osteosarcomaline and with LCLs into which SAP was introduced substantiatedthe role of SAP in the sensitivity to DNA damage in that they diedmore promptly.

In these experiments, however, the results in the comparison ofLCLs derived from healthy donors and XLP patients and thereconstitution of SAP into the XLP-derived LCL differed. In theformer, the presence of SAP led to accumulation of cells in G2/Mphase, although in the latter comparison it led to more pronouncedapoptosis. The difference between these 2 systems may be aconsequence of the difference in the kinetics and amplitude of theresponse to irradiation. In the ‘‘normal’’ LCLs, SAP was induced byp53, although in the reconstituted XLP LCLs, SAP was ectopicallyexpressed, and thus presented from the first moment of theDNA-damage induction.

Altogether, the results consistently indicate that p53 inducedSAP may operate in the elimination of cells with compromisedDNA. The introduction of double-strand breaks into DNA triggers acomplex set of responses, such as cell cycle arrest, relocalization ofDNA repair factors, and apoptosis. Failure to induce these re-sponses in cells with DNA damage can increase the probabilities foroncogenic transformation. We can therefore conclude that the lackof SAP function may allow survival/proliferation of lymphocytesthat carry mutations, cells that may eventually become malignant.

We have thus provided evidence that SAP participates in apo-ptosis. Furthermore, our results suggest that by binding to SAP,VCP may be involved in the regulation of this pathway. VCP (alsocalled p97) is ubiquitous, essential, and abundantly expressed incells. It has an unusually wide variety of functions. Among others,VCP has an anti-apoptotic function as it has been proven indifferent systems. For example, in MCF7 cells, VCP was shown tobe a target of Akt; thus, it is required for cell survival. Its silencingwith siRNA had a potent proapoptotic effect (27). Moreover,studies in yeast showed that a mutant Cdc48 (S565G), that is ahighly conserved yeast orthologue of human VCP, caused mito-chondrial dysfunction, accumulation of cytochrome c in the cytosol,caspase activation, and apoptosis (28). In addition, it has beenshown that VCP is involved in the DNA-damage response pathway(29). These previous findings suggest that the demonstration ofSAP binding to VCP has a biological significance and may be thecritical mediator whereby SAP exerts its apoptotic function. It isconceivable that SAP can preclude VCP from exerting its anti-apoptotic function, a net result being a proapoptotic response of thecells that express SAP.

WehavethusidentifiedSAPasaproapoptoticmoleculebeingreg-ulated by p53. It may work through inhibition of the anti-apoptoticfunction of VCP. With regard to the XLP pathology, we propose thefollowing scenario. In the absence of SAP function, AICD of T cells(homeostasis) is impaired, leading to their uncontrolled prolifera-tion, seen in fatal IM, and DNA damaged lymphoid cells, whichwould normally die, might escape death. If some of these cells wouldstill be able to divide, they might carry mutations and chromosomalaberrations that could contribute to lymphoma development.

Materials and MethodsCell Lines and Treatments. Nadia, LS, TR, and K001-BK are lymphoblastoid cellslines (LCLs). The last 2 LCLs were generated from XLP patients. TR has a complete

Fig. 7. SAP binds VCP, an anti-apoptotic protein. (A) CCRF-HSB2 cells (endog-enous SAP) and Saos-2 cells (transfected SAP) were immunoprecipitated withanti-SAP antibody. The precipitates were probed in a western blot with anti-VCPantibody. (B) Saos-2 cells were transfected with Myc-VCP and SAP plasmids.Forty-eight h later, the cells were immunoprecipitated with anti-SAP antibodyand then the precipitates were probed with anti-Myc.

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deletion of SAP gene, while K001-BK has deletion of exon 2 (26). Saos-2 is ap53-null osteosarcoma cell line. CCRF-HSB2-vector and -SAP cells were activatedwith the indicated concentrations of PMA� ionomycin or 5 �g/mL PHA. Cellswere counted using erythrosin B (Sigma) for detection of dead cells. For testingthe sensitivity of clones to anti-CD95, the cells were treated with anti-APO-1 andprotein A (Sigma). Annexin V-positive cells were detected by flow cytometryusing the annexin V-FITC detection kit (BD PharMingen) following the manufac-turer’s protocol.

DNA-Damage Induction and Cell Cycle Analysis. Gamma irradiation was per-formed in a CIS IBL 637. After the indicated times, cells were collected, washedand resuspended in 0.5 mL PBS, and 1 mL 99% ice-cold ethanol was added.Samples were kept at 4 °C until next day when after washing with PBS, 1 mL of0.1%TritonX-100,20�g/mLpropidiumiodide ,and50�g/mLRNaseAwasadded.After a 20-min incubation at 37 °C, samples were run on a Becton DickinsonFACScan flow cytometer (Becton Dickinson). The CellQuest Plus software (BectonDickinson) was used both for acquisition and analysis of the samples.

Separation and Activation of Primary T Cells. Peripheral blood mononuclear cells(PBMCs)wereseparatedfrombuffycoatsofhealthyblooddonorsbyFicoll-Paque(Amersham Pharmacia). T cells were obtained by negative selection. Cells werecultured in medium alone or in the presence of 1 �g/mL PHA. On the indicatedtime points, 0.1 � 106 cells were plated in 200 �L medium in a 96-well plate. One�Ci 3H-thymidine was added to each well and incubated at 37°C in 5% CO2 for16 h. The cells were harvested on a glass fiber filter, and radioactivity wasmeasuredina liquidscintillationcounter.Alldatarepresentthemeanvaluegivenby 5 parallel wells.

Immunoblotting. The cells were lysed in loading buffer and aliquots correspond-ing to 2 � 105 cells were loaded on each lane. Immunoblotting has been carriedout as described in ref. 14. For detection of different proteins the following Abswere used: a rabbit anti-SAP serum (kind gift of J. Sumegi); DO-7 mAb: p53; mAbClone 124: Bcl-2 (DAKO, A/S), goat polyclonal N-12: VCP (Santa Cruz); 9E10 mAb:Myc-tagged VCP (Santa Cruz). As a control for equal amounts of protein loaded,we detected �-actin by mAb Clone AC-15 (Sigma).

Generation of SAP-expressing Retroviral Vector and Transduction of TargetCells. A retroviral vector, pLXPOP was used to transduce the SAP gene in targetcell lines. The pLXPOP was constructed from the pLNPOX vector (30). SAP cDNAwas amplified by PCR from a pcDNA3 plasmid carrying the SAP gene (kind giftfrom Dr. Janos Sumegi, Cincinnati Children’s Hospital Medical Center, OH). ThePCR product was cloned into the retroviral vector. The insert was later sequenced.Retrovirus particles were produced in the Phoenix cell line. The virus containingsupernatant was collected, filtered through 0.45-�m HT Tuffryn membrane filter

(Pall Corporation), and polibrene (Sigma) was added to a final concentration of4 �g/mL. Thirty-six hours after infection of the target cells with this supernatant,selection of transduced cells was started with puromycin.

Expression Vectors and Transfections. The VCP cDNA was amplified from thepCMV-SPORT6 construct carrying the BC110913.1 clone sequence of VCP (RZPDGerman Resource Center for Genome Research). The sequence was then clonedinto the pCMV-Myc vector (Clontech) and the insert was sequenced. Vectors weretransfected into Saos-2 cells in 10-cm Petri dish by polyethyleneimine (PEI) tech-nique. Forty-eight hours after transfection, the cells were collected, lysed, andsubjected to coimmunoprecipitations.

Coimmunoprecipitations. Cells were lysed in cell lysis buffer (50 mM Tris, pH 7.6;150 mM NaCl; 1% Nonidet P-40; 0.5% sodium deoxicholate; and 0.05% SDS)containing protease inhibitors mixture (Complete Mini, Roche) for 30 min on ice.Insoluble material was removed by centrifugation at 14,000 � g for 15 min.Protein G-sepharose precleared lysates were incubated with rabbit anti-SAP ormouse anti-Myc antibodies overnight at 4 °C. As control, equal volumes of lysateswere incubated with normal rabbit serum or with normal mouse serum. ProteinG-sepharose was added again for 2 h. The precipitates were washed 5 times withPBS containing protease inhibitor mixture. Affinity-bound protein complexeswere released from protein-G beads by boiling in loading buffer and analyzed bySDS-PAGE.

Silver Staining and Mass Spectrometry. Immunoprecipitated samples weresubjected to reduction and alkylation by pretreating them with DTT and iodoac-etamide. After running them on SDS-PAGE, proteins were visualized by silverstaining (31). Relevant bands were excised and treated for in-gel digestion (32).In brief, the silver stained bands were destained, dried and trypsin (PromegaCorporation) was allowed to soak into the gel over ice. After incubation at 30 °Covernight, the samples were acidified and the generated peptides were concen-trated and desalted on a �C18 ZipTip (Millipore). The peptide mixture was elutedwith 70% ACN containing the matrix alfa-cyano-4-hydroxy-cinnamic acid directlyonto the MALDI target. Spectra for peptide mass fingerprinting were obtainedonaMALDI-TOF/TOFmass spectrometer (Ultraflex III,BrukerDaltonics) followingthe manufacturer’s recommendation. Spectra were internally calibrated usingautolytic peptides from trypsin resulting in an error �15 ppm. Search for identitywas done via ProFound (http://prowl.rockefeller.edu). The searches were donetotally un-biased, that is, no species, size or pI was specified.

ACKNOWLEDGMENTS. The authors thank Dr Janos Sumegi for generouslyproviding cell lines and plasmids. This work was supported by funds from theSwedish Cancer Society, Sweden. L.L.K. is a recipient of a Cancer Research Fel-lowship of Cancer Research Institute (New York)/Concern Foundation (Los An-geles).

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