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Adenovirus infection and cytotoxicity of primary mantle cell lymphoma cells
Experimental Hematology 33 (2005) 1337–1347
Adenovirus infection and cytotoxicity of primary mantle cell lymphoma cells Daniel J. Medinaa,b, Wendy Sheaya, Mona Osmanc, Lauri Goodellc, John Martina, Arnold B. Rabsona,d,e, and Roger K. Straira,b a
The Cancer Institute of New Jersey and Departments of bMedicine, cPathology, and dMolecular Genetics and Microbiology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, N.J., USA; e Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey, Piscataway, N.J., USA (Received 10 February 2005; revised 29 June 2005; accepted 11 July 2005)
Mantle cell lymphoma (MCL) is a distinct form of non-Hodgkin’s lymphoma (NHL) derived from CD5+ B cells. MCL cells overexpress cyclin D1 as a consequence of translocation of the gene into the immunoglobulin heavy-chain gene locus. MCL is an aggressive form of NHL with frequent relapses after standard-dose chemotherapy. In this context, a variety of novel therapies for patients with MCL have been investigated. In this study, we use an expanded panel of attenuated adenoviruses to study adenovirus-mediated cytotoxicity of MCL cells. Our results demonstrate: 1) adenovirus infection of MCL cells despite the absence of receptor/coreceptor molecules known to be important for adenovirus infection of other cells types; 2) cytotoxicity of MCL cells after infection with specific adenovirus mutants; 3) a high degree of cytotoxicity after infection of some patient samples with viruses lacking the E1B 19k ‘‘antiapoptotic’’ gene; and 4) cytotoxicity after infection with viruses containing mutations in E1A pRb or p300 binding. The extent of cytotoxicity with the panel of viruses demonstrated interpatient variability, but 100% cytotoxicity, as determined by molecular analysis, was detected in some samples. These studies provide the foundation for: 1) the development of adenoviruses as cytotoxic agents for MCL and 2) analyses of key regulatory pathways operative in MCL cells. Ó 2005 International Society for Experimental Hematology. Published by Elsevier Inc.
Mantle cell lymphoma (MCL) is a distinct subtype of nonHodgkin’s lymphoma (NHL) derived from CD5D B cells originating from the mantle zone surrounding reactive germinal centers. MCL accounts for 5 to 8% of adult NHL in the United States and Europe. The disease is aggressive with a median patient survival of 2 to 4 years. Although standard chemotherapeutic regimens generally induce disease response, the responses are often partial and of short duration. Even when a complete remission is obtained, the median duration is short with no plateau in failure-free survival [1–7]. Improved outcomes occur with more intensive regimens with or without autologous or allogeneic hematopoietic stem cell rescue/transplantation; however, many patients still relapse and these intensive therapies are difficult to administer, are associated with many adverse effects, and are often limited to younger patients. Hence, MCL is an ideal disease for the development of novel therapeutic agents.
Offprint requests to: Daniel J. Medina, Ph.D., The Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901; E-mail: [email protected]
MCL is characterized by the proliferation of CD5D, CD19D, CD232 monoclonal B cells containing the chromosomal translocation t(11;14) (q13;q32), which places the cyclin D1 gene (BCL-1/CCND1/PRAD-1) under the transcriptional control of immunoglobulin heavy-chain enhancer elements [8–10]. The resulting overexpression of cyclin D1 is thought to play a key role in the pathogenesis of MCL. Cyclin D1 is important in regulating the G1-to-S phase transition of the cell cycle [11]. Cyclin D1 interacts with cyclin-dependent kinases (Cdk) 4 and 6 to form a complex that phosphorylates the retinoblastoma protein (pRb), causing the release of E2F transcription factors, which are essential for the expression of S-phase genes. Recent studies indicate that loss of function mutations/deletions in cell cycle regulatory proteins, ataxia telangiectasa mutated (ATM), and altered expression of apoptotic regulatory proteins expression also contribute to the development of MCL [10,12–16]. Therefore, multiple genetic events targeting cell cycle, response to DNA damage, and induction of apoptosis contribute to mantle cell lymphomagenesis. The genetic alterations that mediate cell cycle progression, repression of cellular response to DNA damage and inhibition of apoptosis in MCL cells, are analogous to
0301-472X/05 $–see front matter. Copyright Ó 2005 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 10.1016/j.exphem.2005.07.009
D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
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virus-induced changes that occur during adenovirus, human papillomavirus, or polyomavirus infection. For example, during a productive adenovirus infection, the virus induces cellular pathways that support cell cycle progression and DNA replication while suppressing DNA repair and apoptosis. This strategy allows cellular support of viral DNA synthesis while allowing the cell to survive long enough to produce progeny virus. To achieve cell cycle progression without induction of apoptosis, adenovirus gene products interact with many of the same strategic regulatory pathways that are altered in malignant cells such as MCL cells (Fig. 1). For example, adenovirus gene products target p53, pRb, p300, DNA damage pathways, and apoptotic pathways. Hence there may be redundant targeting of key pathways when an adenovirus infects a malignant cell [17–20]. As a result, attenuated/mutated adenoviruses may be constructed to infect and conditionally replicate or induce cytotoxicity in tumor cells, as previously demonstrated for malignant cells with p53 mutations [17,18]. In those studies, attenuated adenoviruses containing a mutation that prevented inactivation of p53 selectively killed p53-deficient epithelial cells. In this study,
we demonstrate adenovirus infection of primary MCL cells and use a panel of attenuated adenoviruses to infect and kill MCL cells. This study was prompted by the knowledge that multiple perturbations of several important cellular pathways in MCL may provide a cellular milieu that complements specific adenovirus mutations, allowing the development of mutant viruses with selective cytotoxicity. For example, adenovirus early gene products encoded by the E1 region (E1A and E1B) are expressed shortly after infection. E1A encodes two major overlapping polypeptides, a 286 amino acid polypeptide and a 243 amino acid polypeptide. The 286 polypeptide contains a functional domain, complementary region 3 (CR3), that regulates viral gene expression. In addition, both of the E1A polypeptides interact with a variety of host transcription and chromatin remodeling factors, including pRb and p300. The other E1 region, E1B, also encodes key regulatory proteins. The E1B 55-kd polypeptide interacts with p53 and the E1B 19-kb polypeptide inhibits apoptosis [17–20]. As these E1 gene products modulate key cellular pathways such as pRb and p53 that are altered in MCL cells, we hypothesized that specific viral
CYCLIN D1
S phase viral gene expression
E2F p300 pRb
E1A
Early gene expression CR 3 pRb
p300 binding
orf 4
E4 induction
apoptosis
orf 3/6 binding
p14 arf
E1B19K
antiapoptotic
E1B55K
p53
altered p53-mdm2 interaction
ATM cell cycle arrest
p53
bax, CD95
bcl-2 IAP apoptosis
cellular stress altered transcriptional co-activation
mdm2
Figure 1. A simplified model of adenovirus protein–MCL cell interactions. Adenovirus E1A and E1B are expressed early during infection. E1A complementary region (CR) 1 and 2 are included on both a large (286 aa) and small (243 aa) polypeptide. CR3 is unique to the large E1A polypeptide and activates adenovirus early gene expression. The E1A pRb and p300 binding sites are present in both polypeptides. E1A p300 binding may impact on cytotoxicity by modulating host and viral transcription, increasing p53 (impairment of mdm2 induction or alteration of mdm2-p53 interactions) and forming a complex with pRb. E1A pRb binding induces E2F family members (as well as other proteins), resulting in induction of several genes required for G1-S transition and adenovirus DNA synthesis. p14arf is also induced by E1A and may enhance p53 stability. Stabilized p53 may result in proapoptotic bax or CD95 expression. To counteract the proapoptotic effects of p53 expression, the E1B region encodes: 1) a 55K protein that binds p53 and collaborates with E4-encoded peptides (open reading frames [orf]3/6) to transport and degrade p53 and 2) a 19k protein that has potent antiapoptotic activity. Other early proteins encoded by E4 may modulate apoptosis by binding to cellular phosphatases and/or inhibiting DNA repair complexes induced by viral DNA. Cyclin D1 overexpression in MCL may result in many biological effects, including pRb hyperphosphorylation, which could complement E1A pRb binding.
D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
mutants, such as those that alter E1A ‘‘inactivation’’ of pRb, might be complemented by cyclin D1 overexpression and result in selective cytotoxicity. In this study we tested the above hypothesis and demonstrate that infection of MCL cells with a panel of mutated adenoviruses, including those with specific mutations in E1A, results in cytotoxicity. Furthermore, we demonstrate infection of MCL cells via a novel coxsackie-adenovirus receptor (CAR)-independent pathway not observed in previous studies with chronic lymphocytic leukemia (CLL) and multiple myeloma [19,20]. These studies provide preclinical information that will foster the development of attenuated adenoviruses as therapeutic agents for patients with MCL and also provide the foundation for future molecular analyses to define the dynamic activity of specific key regulatory pathways in MCL. Material and methods Patients Six MCL patients (samples MCL-1–MCL-6) were included in this study. Diagnosis was based on immunophenotype (CD5D, CD19D, and CD232) of malignant cells in conjunction with expression of cyclin D1 and/or detection of the translocation t(11;14) by cytogenetics or fluorescence in situ hybridization analysis. Samples of blood or spleen were obtained under a protocol approved by the Robert Wood Johnson Medical School Institutional Review Board. Mononuclear cells were isolated from 10 to 25 mL of fresh heparinized blood or splenectomy sample by Histopaque centrifugation. Isolated cells were counted, resuspended in freezing medium (90% fetal bovine serum and 10% dimethylsulfoxide), and stored in liquid nitrogen until use. Analyzed MCL cells obtained from blood were greater than 95% MCL cells as determined by flow cytometry (MCL-1, 97%; MCL-2, 95%; MCL-3, 97%; MCL-4, 98%; MCL-5, 96%; MCL-6, 97%). The sample obtained from the spleen of patient MCL-1 was 75% MCL cells, as determined by flow cytometry. Cells and viruses Two hundred ninety-three cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with antibiotics, 2 mM L-glutamine, and 10% fetal bovine serum (FBS). MCL cells were maintained ex vivo in RPMI 1640 medium supplemented with antibiotics, 2 mM L-glutamine, and 10% FBS. The viruses used in the experiments, Ad5dl309, E1A-, Pac3, and Ad5dl337, were described previously [19–22]. The wild-type parent for these mutant viruses, Ad5dl309, lacks a portion of the E3 region that has been shown to be dispensable for growth in cell culture [23]. The following E1A mutant adenoviruses were generously provided by Elizabeth Moran (Temple University, Philadelphia, PA, USA): 12S.RG2, E1A.RG2, 12Swt, 12S.YH47, and E1A.YH47 [24]. All viruses were grown and titered on 293 cells at 37 C. For mock infections, Ad5dl309 was heat-inactivated at 65 C for 30 minutes. The recombinant adenovirus containing the green fluorescent protein (GFP) was purchased from Quantum Biotechnologies (Montreal, Quebec, Canada). That virus (AdGFP) contains the GFP gene under the control of a cytomegalovirus (CMV) promoter, in the E1 region of adenovirus. A summary of the viruses is presented in Table 1.
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Table 1. Viruses used in this study
Virus Ad5 dl309 Ad5 E1A2 Pac3 Ad5 dl337 E1A 12S E1A YH47 928 E1A RG2 12S YH47 928 12S RG2
E1A p300
E1A pRb
E1A CR3
E1B 55K
E1B 19K
D 2 2 D D D 2 D 2
D 2 2 D D 2 D 2 D
D 2 2 D 2 D D 2 2
D D 2 D D D D D D
D D 2 2 D D D D D
Infection MCL cells (2 3 106) were infected with the indicated viruses at a multiplicity of infection (moi) of 100 for 4 to 6 hours at 37 C. After infection, the cells were washed three times with Dulbecco’s phosphate-buffered saline to remove nonadsorbed virus. The cells were then pelleted by centrifugation at 500g for 10 minutes, resuspended in 2 mL of medium, and culture in 24-well plates. At the indicated times, samples were removed for determination of cell viability by trypan blue staining and virus yield. Heat-inactivated Ad5dl309 were used for mock infections. Virus assay Cell-free supernatants were collected at the indicated times to assess virus production. The virus titer was determined by plaque assay on 293 cells. Briefly, 1 mL of serial 10-fold dilutions of cellfree supernatant were added to duplicate 60-mm dishes containing a 95% confluent layer of 293 cells for 1 hour at 37 C. After incubation, the supernatant was removed and the plates were overlaid with agarose as described [25]. Viral plaques were counted on day 14 and the viral titer was determined Flow cytometric analysis for CAR and integrin expression Cell surface expression of CAR was determined by indirect immunofluorescence flow cytometry. The primary monoclonal antibody (mAb) RcmB anti-CAR (provided by Robert Finberg Dana-Farber Cancer Institute, Boston, MA, USA) was used at a 1:100 dilution and visualized using fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (Sigma, St. Louis, MO, USA) secondary antibody at 1:200 dilution. Cell surface expression of integrins was determined by flow cytometry using FITC-conjugated antibodies (1 mg/mL) for avb3, avb5, b1, and aM (Chemicon, Temecula, CA, USA). In every experiment an irrelevant isotype-controlled mAb was used to determine background staining. All staining procedures were for 30 minutes at 4 C in Hanks balanced salt solution (HBSS) that contained 5% FBS. Flow cytometric analysis was performed using a FACScan analyzer. Immunoglobulin heavy-chain analysis To assess molecular responses after adenovirus infection of MCL cells, primary cells from the spleen of patient MCL-1 were infected with Ad5dl309 at an moi of 100 and washed three times with PBS. Mock (heat-inactivated Ad5dl309) infection of MCL cells was used as a control. At the indicated time, 2 3 104 cells from each of the infections was mixed with 3 3 106 uninfected Jurkat T cells (as a carrier). Viable cells were obtained by Ficoll-hypaque density separation and the isolated DNA was
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D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
polymerase chain reaction (PCR) amplified with allele-specific primers for patient MCL-1 [26]. Additional controls included DNA isolated from Jurkat cells, uninfected MCL-1 cells, and cells from another patient with MCL. This last control was used to demonstrate the specificity of the allele-specific primers. Statistics The mock infection was treated as the control group. Therefore, each specific virus was analyzed independently at each time point versus the mock infection using Student’s t-tests, at the 0.05 level of significance. Further analyses were completed using a one-way analysis of variance, at the 0.05 level of significance, to determine the statistical differences between baseline and all subsequent time points postinfection for each of the viruses and the mock infection. Analyses were completed using Microsoft Excel 2000 (Redmond, WA, USA) and SPSS Version 11.5 (Chicago, IL, USA).
Results Infection of primary MCL cells with AdGFP Initial experiments established the capacity of adenoviruses to infect MCL cells. In these studies, a recombinant
MCL-1
adenovirus expressing GFP under the control of a CMV promoter (AdGFP) was used to infect MCL cells. Five days after infection, the number of cells expressing green fluorescence was measured by flow cytometry. In each MCL sample a marked shift in fluorescence was observed after infection with AdGFP (Fig. 2). In all cases, the mean intensity of the entire population shifted in a statistically significant fashion, indicating infection of a large percentage of cells. In contrast, as previously described, neither normal B cells obtained from tonsil infected with AdGFP [19] nor MCL cells infected with a control virus not containing the GFP gene (Fig. 2) demonstrated any statistically significant shift in intensity after infection. Cell surface expression of CAR and integrins on MCL cells In permissive cells, adenovirus infection is mediated by the quantity of both CAR and av integrins expressed on the surface of the target cells [27–30]. Lymphocytes, however, are generally resistant to adenovirus infection, in part because they normally lack expression of CAR and/or av integrins [31–34]. Because MCL cells from different patients showed variable infection efficiency with AdGFP, we
MCL-2
MFI P=0.01
MCL-4 MFI P=0.04
MCL-6
MCL-4 (Pac3)
MCL-5 MFI P=0.02
MFI P=0.02
MFI P=0.01
MFI P=0.29
CLL
Figure 2. MCL cells can be infected with an adenovirus encoding GFP. MCL cells were infected with AdGFP (moi 5 100), containing the GFP gene under control of a CMV promoter. Five days after infection, cells were assayed for GFP expression by flow cytometry. The bold line represents the fluorescence of AdGFP-infected cells. The lighter line histogram represents the fluorescence of uninfected cells. The panel labeled MCL-4 (Pac3) represents a control infection of MCL cells with an adenovirus not expressing GFP. The panel labeled CLL represents AdGFP infection of CLL cells and was used as a positive control, as described by Medina et al. [19]. Statistical analysis of the shift in mean fluorescence intensity after infection is indicated in the upper right corner of each panel.
D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
characterized the expression of CAR and integrins avbb3, avb5, b1, and aM on MCL cells from five patients, as well as normal tonsil derived B cells by flow cytometry. The results are summarized in Table 2 and a representative histogram (MCL-4) is shown in Figure 3. As shown in Table 2, tonsil-derived B cells as well as cells MCL-1, MCL-2, MCL-4, and MCL-5 did not express CAR or avb3, avb5, and aM. MCL-6 expressed only low levels of CAR and was also negative for avb3, avb5, and aM expression. Only b1 integrin was consistently expressed at high levels in both tonsil-derived B cells and MCL cells. In contrast, high-level CAR expression has been detected on CLL cells [20,34]. These data suggest that alternate receptors may mediate adenovirus infection of MCL cells. Replication-competent adenovirus (Ad5dl309) is cytotoxic to MCL cells To determine whether adenoviruses induce cytotoxicity after infection of MCL cells, MCL-1 cells were infected with the parental virus Ad5dl309 or Ad5dl309 mutants containing deletions of either E1A (E1A2) or deletions of E1A and E1B (Pac3; see Table 1). This population of MCL cells was prepared from spleen and contained w75 % MCL cells. Cell viability was monitored over the course of 10 days by trypan blue exclusion. After 10 days in culture, heat-inactivated Ad5dl309-infected cells (mock-infected control), E1A2 virus-infected cells, and Pac3-infected cells had no loss of cell viability. In contrast, cytotoxicity was detected after Ad5dl309 infection, with viability reduced by 80% compared to control cells (Fig. 4). To determine whether residual viable MCL cells remained after 10 days of Ad5dl309 infection of MCL-1 cells, cellular DNA from viable mock-infected and Ad5dl309-infected cells were assayed for allele-specific immunoglobulin H (IgH) rearrangement by PCR using patient-specific primers. Mock-infected MCL-1 cells remained positive for the allelespecific IgH rearrangement while MCL-1 cells infected with Ad5dl309 demonstrated complete eradication of a PCR signal corresponding to the allele-specific IgH rearrangement (Fig. 5). Although only a qualitative PCR assay was performed, limiting dilution experiments (not shown) indicated
Table 2. CAR and integrin expression on MCL cells and tonsil B cells* Patient
CAR
b1
aM
aVb3
aVb5
Tonsil-1 MCL-1 MCL-2 MCL-3 MCL-4 MCL-5 MCL-6
4.6 0.1 2.8 ND 5.5 0.6 15
34.2 50.1 79.6 ND 10.8 83.1 97.2
6.5 1.4 1.0 ND 1.6 1.8 1.8
8.2 4.3 5.2 ND 10.4 5.1 5.1
2.3 1.2 3.3 ND 2.2 1.3 5.3
*Expressed as the % of cells staining positive with the indicated FITClabeled antibody compared to FITC-labeled isotype control.
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that Ad5dl309 infection resulted in at least a 3 log reduction in MCL cells. Mutated adenoviruses are cytotoxic to MCL cells Previous studies from our laboratory examining adenovirus-mediated cytotoxicity after infection of primary CLL cells demonstrated high patient variability with regards to cytotoxic effects induced by a given virus [19]. Therefore, we examined the use of attenuated adenoviruses in five additional MCL patients. In this experiment, MCL cells were infected with Ad5dl309, E1A2, Pac3, or an Ad5dl309 mutant containing a deletion in the E1B 19k gene (Ad5dl337; see Table 1). The cells were infected as described above and the cytopathic effects of the viruses were evaluated at the indicated time points by trypan blue exclusion. Nine to 12 days after infection with Ad5dl309, MCL-2, MCL3, MCL-4, MCL-5, and MCL-6 cells exhibited 65 to 80% cytotoxicity (Fig. 6). Infection with adenovirus deleted in both E1A and E1B (Pac3) resulted in minimal cytotoxicity (10–30%) after infection of MCL-2, MCL-4, MCL-5, and MCL-6. However, MCL-3 cells demonstrated O50% cytotoxicity on day 9. E1B 19K2 (Ad5dl337) resulted in marked cytotoxicity after infection of most of the samples, with near complete cytotoxicity of MCL-2 cells by day 12. MCL cells infected with the E1A2 adenovirus also demonstrated variable cytotoxicity. MCL-2 and MCL-3 demonstrated 75% cytotoxicity after infection with the E1A2 virus, whereas MCL-1, MCL-4, and MCL-6 demonstrated lesser degrees of cytotoxicity after infection with E1A2. Infection of MCL cells with adenoviruses containing E1A p300-binding and E1A pRb–binding mutations As a result of the t(11;14) translocation, MCL cells overexpress cyclin D1, which may result in phosphorylation of pRb and complement adenovirus E1A pRb binding. To begin to assess the ability of cyclin D1 to complement E1A activity, viruses with specific mutations in E1A were studied. These viruses had mutations in E1A p300–binding (RG2) or pRb-binding (YH47) sites in the context of either full E1A (13S) or a CR3-deleted E1A (12S; see Table 1). Adenoviruses containing the full E1A gene (13S) were cytotoxic, and cytotoxicity was maintained by individual mutations of either the E1A p300– or pRb-binding site (Fig. 7). However, in all patient samples for which there were enough cells to perform three independent infections with each virus (i.e., MCL-2, MCL-3, MCL-4, MCL-6), E1A.RG2 (with the p300-binding mutation) demonstrated statistically greater cytotoxicity ( p ! 0.05) than E1A.YH47 (with the pRb-binding mutation). When studied in the context of viruses containing deletion of E1A CR3 (12S.RG2 versus 12S.YH47, see Table 1), three of the four samples (MCL-3, MCL-4, MCL-6) had statistically greater cytotoxicity ( p ! 0.05) after infection with the virus containing p300-binding mutation. In the fourth sample (MCL2), the reverse pattern was seen after infection with
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D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
Figure 3. Cell surface expression of CAR and integrins on MCL cells. MCL cells were labeled with the respective monoclonal antibodies and analyzed by flow cytometry. The open histogram represents staining after reaction with an isotype control antibody. The shaded histogram represents staining with antibodies to adenovirus receptor CAR and the integrins b1, aM, avb3, and avb5.
12S.RG2 and 12S.YH47. The discrepancy in cytotoxicity after infection of MCL-2 (enhanced killing with viruses containing p300 mutation in the context of a full-length virus but decreased killing with viruses containing p300 mutation in the context of the 12S virus) may reflect the genetic heterogeneity of MCL and the complexity of virus–host interactions in the setting of these interpatient genetic differences. These data suggest that in several patient samples and virus backgrounds viruses that contain mutations that inhibit E1A-pRb binding are not fully complemented by cyclin D1 overexpression in MCL cells.
Discussion Previous work by others has established the capacity of adenovirus mutants to selectively infect and kill malignant epithelial cells containing p53 mutations [17,18]. The intent of this study was to determine whether adenoviruses with specific cytotoxicity for MCL cells could be developed. In our initial studies, MCL and CLL cells, but not tonsilderived B cells, were infected with adenovirus. MCL cells from different patients demonstrated variable infection efficiency. To determine whether some of this variation was due to different expression levels of cell surface molecules
D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
MCL-1 140
% of Control
120 100 80 60 40
*
20 0
Mock
dl309
E1A
Pac3
Figure 4. Adenovirus infection of MCL cells induces cytotoxicity. Cells from the spleen of patient MCL-1 were infected with Ad5dl309, mock-infected (heat-inactivated Ad5dl309), an E1A-deleted adenovirus (E1A2), or an adenovirus with a deletion of E1A and E1B (Pac3). At the indicated time cell viability was determined by trypan blue staining and compared to the viability of uninfected control cells. The asterisk indicates a statistically significant ( p ! 0.05) difference in cytotoxicity after infection compared to mock-infected control.
known to function as adenovirus receptors and coreceptors [27–30], we evaluated the expression of CAR and the coreceptors av integrins on MCL cells. Normal B cells were not efficiently infected by adenoviruses and had no/low expression of both CAR and avb3, avb5, and aM integrins [19,20]. In contrast, CLL cells, which can be infected by adenoviruses, uniformly expressed the CAR receptor and avb3, avb5 integrins but not aM integrins [20]. Surprisingly, MCL cells were infected at a level comparable to that seen for CLL cells but expressed undetectable or minimally detectable levels of CAR and coreceptors avb3, avb5, and aM integrins. These data suggest that adenovirus entry into
std
1
2
3
4
5
6
Figure 5. Molecular (PCR) analysis of adenovirus-mediated cytotoxicity of MCL cells. MCL cells from patient 1 were infected with Ad5dl309 or mock-infected with heat-inactivated Ad5dl309. The cells were maintained for 10 days and the surviving cells were assayed by qualitative IgH allele-specific PCR. Lanes: std, marker; 1, H2O; 2, Jurkat cell DNA; 3, DNA from MCL-1 infected with Ad5dl309; 4, DNA from MCL-1 mock-infected; 5, DNA from patient MCL-2; 6, DNA from uninfected MCL-1 cells.
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MCL cells is mediated by pathways distinct from those operative in CLL cells and independent of CAR and/or avb3, avb3, and aM integrins. Therefore, CAR expression cannot be used as a surrogate for determining the capacity of the cell to be infected by adenoviruses and novel adenovirus ‘‘receptors’’ may be present on MCL cells. Studies are currently underway to elucidate the mechanisms associated with adenovirus binding/internalization in MCL cells. In addition, because CLL cells, but not MCL cells, express high levels of CAR we are evaluating the absence of CAR expression as a distinguishing immunophenotypic characteristic of MCL (in comparison to other lymphoproliferative disorders). Having established that adenoviruses infect primary MCL cells, infection with a panel of adenovirus mutants was undertaken to determine their spectrum of cytotoxicity in individual patient samples. These studies demonstrated that attenuated adenoviruses are capable of inducing cytotoxicity in MCL cells and that the degree of cytotoxicity induced by individual viruses varied among MCL samples. This patient variation in adenovirus-induced cytotoxicity with individual viruses is similar to that reported for adenovirus infection of CLL cells [19] and is most likely the result of genetic variation in the individual patient malignant cells. Of the viruses examined in the first set of infections, the replication-competent Ad5dl309 and the mutated Ad5dl337 (E1B 19K deletion) had the broadest range and extent of cytotoxicity. The most attenuated virus, Pac3 (deletion of E1A and E1B), induced the least toxicity in most samples. The adenovirus deleted in E1A (E1A2) demonstrated variability in terms of cytotoxicity. Hence, E1A activity is not uniformly and completely complemented by cyclin D1 overexpression in MCL cells. This lack of full E1A complementation by cyclin D1 overexpression likely reflects the multiple functions and binding partners of E1A during the viral life cycle [35–39]. To refine the studies of adenovirus E1A mutant viruses, attenuated viruses with mutations in either pRb or p300 binding were studied. As cyclin D1 binds and phosphorylates pRb, facilitating E2F-1 release and cell cycle progression, it was hypothesized that an attenuated adenovirus containing a mutation in E1A pRb binding might be fully complemented in MCL cells. Although cytotoxicity was seen after infection with this E1A pRb binding mutant virus, the extent of cell killing was reduced in comparison to infection with a virus containing intact E1A or, in several patient samples, a virus with an E1A mutation in p300 binding. Therefore, E1A pRb binding is not fully complemented by overexpression of cyclin D1. The relative lack of E1A-pRb-binding complementation in MCL cells may relate to a lack of complete pRb phosphorylation by cyclin D1 or to more complex interactions between cyclin D1, E1A, and pRb. For example, hyperphosphorylation of pRb and increased E2F-1 expression has been detected in a subset of patients with MCL, but
D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
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MCL-2 120
*
*
*
*
80 60 40
100
20
80
* *
40
0
4
8
3
12
Mock dl309 E1APac3 dl337
120
% of Control
* *
*
60
*
*
*
40
0
3
100 80 60 40
0
9
6
3
Days
MCL-6
*
*
6
9
Days
120
% of Control
Mock dl309 E1APac3 dl337
20
20
100
9
MCL-5
120
% of Control
MCL-4
*
6
Days
Days
80
*
*
60
20
0
100
Mock dl309 E1APac3 dl337
120
% of Control
% of Control
100
MCL-3
Mock dl309 E1APac3 dl337
*
Mock dl309 E1APac3 dl337
*
80
*
*
60 40 20 0 3
6
9
Days Figure 6. Attenuated adenovirus infection of MCL cells induces cytotoxicity. Primary MCL cells from five patients were infected with Ad5dl309, heat-inactivated Ad5dl309 (Mock), an E1A-deleted adenovirus (E1A2), an adenovirus with a deletion of E1A and E1B (Pac3), or an adenovirus containing an E1B 19K deletion (Ad5dl337). At the indicated times, cell viability was determined by trypan blue staining and compared with the viability of uninfected cells. Asterisks indicate statistically significant differences in cytotoxicity ( p ! 0.05) compared to mock-infected controls.
there is no clear correlation of pRb phosphorylation with the degree of cyclin D1 overexpression [15,16]. In fact, there are many other biological activities of cyclin D1 that might contribute to oncogenesis. For example, pRb interaction without phosphorylation, cyclin D1-induced transcriptional modulation, cyclin D1 sequestration of cyclin-dependent kinases and inhibitors, and/or cyclin D1-mediated antiapoptotic effects have been described [40–45]. Transcriptional modulation via interaction with (and sequestration of) the transcription factor DMP1 may be relevant, as DMP1 is required for the induction of p14arf, a regulator of p53 induction by a variety of oncogenic stimuli, including adenovirus early polypeptides
[45–48]. Hence, there are many potential mechanisms by which cyclin D1 overexpression might contribute to MCL lymphomagenesis in the absence of direct effects on pRb. In fact, overexpression of cyclin D1 and p16INK4A loss can be seen concurrently in MCL, suggesting that there may be additional independent, nonoverlapping mechanisms by which both pRb inactivation and cyclin D1 contribute to MCL generation/maintenance [49]. In addition, mutational analysis of cyclin D1 has demonstrated that the pRb kinase domain is dispensable for cellular transformation in some assays [50]. Hence, cyclin D1 overexpression may mediate oncogenic effects other than pRb phosphorylation.
D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
% of Control
120 100
*
80
*
60
*
40
MCL-3 120 100
% of Control
Mock dl309 12S.RG2 E1A.RG2 12S wt 12S.YH47 E1A.YH47
MCL-2
*
80
4
40
8
3
12
Days Mock dl309 12S.RG2 E1A.RG2 12S wt 12S.YH47 E1A.YH47
% of Control
100
*
* *
40
MCL-5 120 100
% of Control
MCL-4
60
6
10
Days
120
Mock dl309 12S.RG2 E1A.RG2 12S wt 12S.YH47 E1A.YH47
80 60 40 20
20 0
0 3
6
9
MCL-6
120
*
80
3
6
9
Days
Days
% of Control
*
60
0
0
100
*
Mock dl309 12S.RG2 E1A.RG2 12S wt 12S.YH47 E1A.YH47
20
20
80
1345
Mock dl309 12S.RG2 E1A.RG2 12S wt 12S.YH47 E1A.YH47
* *
60 40 20 0 3
6
9
Days Figure 7. Infection of MCL cells with adenoviruses containing E1A-pRb- or E1A-p300-binding mutants induce cytotoxicity. Primary MCL cells from five patients were infected with the indicated viruses at an moi of 100. At the indicated times, cell viability was determined by trypan blue staining and compared to mock-infected cells. The parental virus, Ad5dl309, contains the complete E1A region; E1A.RG2 has a mutation of the E1A-p300 binding site; E1A.YH47 has a mutation of the E1A-pRb binding site. The 12Swt lacks the CR3 but retains the E1A-p300 and -pRb binding sites; 12S.RG2 lacks E1A CR3 and has a mutation of the E1A-p300 binding site; 12S.YH47 lacks E1A CR3 and has a mutation of the E1A-pRb binding site. Asterisks indicate infections that result in statistically significant greater cytotoxicity ( p ! 0.05) than the control (mock-infected) cells. Statistical comparisons of E1A.RG2 and E1A.YH47 infection and 12S.RG2 and 12S.YH47 infections are described in the text.
Much of what is known concerning cyclin D1 and other regulatory proteins involved in the control of cell cycle, apoptosis, stress response, and transcription is based upon molecular analysis of cell lines and transgenic mice. Such studies have provided fundamental information concerning key regulatory interactions, but variable biological effects are often seen in different cell types and contexts. This dependence upon cell type and physiology emphasizes the importance of analyzing basic regulatory protein interactions in primary malignant cells. Studies of adenovirus infection in these cells may allow a dynamic analysis of
regulatory pathways active during infection with selected adenovirus mutants and may identify key regulatory pathways that are active in individual patient MCL cells. The variability in outcomes after infection with different viruses implies an interpatient heterogeneity in the activity of regulatory and/or oncogenic pathways. Studies such as these will hopefully allow these pathways to be better characterized in primary cells and, ultimately, targeted by either attenuated adenoviruses or other directed therapies. In summary, this report demonstrates MCL cell infection with adenoviruses despite absent or very low-level
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expression of CAR or selected integrins known to mediate adenovirus infection in other cells. Adenovirus-mediated cytotoxicity is demonstrated after infection of MCL cells, and there is variable cytotoxicity after infection with viruses attenuated by specific mutation of adenovirus E1 genes. The requirement of E1A-pRb binding for maximum cytotoxicity implies that cyclin D1 expression cannot completely complement E1A-pRb binding under the conditions of the infection. In addition, adenovirus-mediated cytotoxicity after infection of MCL cells with attenuated viruses that do not result in cytotoxicity after infection of normal B cells raises the prospect of developing attenuated adenoviruses for therapeutic use in patients with MCL.
Acknowledgments Supported by Lymphoma Research Foundation and the Virginia L. Koehler Lymphoma Grant Program, The New Jersey Commission on Cancer Research.
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Adenovirus infection and cytotoxicity of primary mantle cell lymphoma cells Daniel J. Medinaa,b, Wendy Sheaya, Mona Osmanc, Lauri Goodellc, John Martina, Arnold B. Rabsona,d,e, and Roger K. Straira,b a
The Cancer Institute of New Jersey and Departments of bMedicine, cPathology, and dMolecular Genetics and Microbiology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, N.J., USA; e Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey, Piscataway, N.J., USA (Received 10 February 2005; revised 29 June 2005; accepted 11 July 2005)
Mantle cell lymphoma (MCL) is a distinct form of non-Hodgkin’s lymphoma (NHL) derived from CD5+ B cells. MCL cells overexpress cyclin D1 as a consequence of translocation of the gene into the immunoglobulin heavy-chain gene locus. MCL is an aggressive form of NHL with frequent relapses after standard-dose chemotherapy. In this context, a variety of novel therapies for patients with MCL have been investigated. In this study, we use an expanded panel of attenuated adenoviruses to study adenovirus-mediated cytotoxicity of MCL cells. Our results demonstrate: 1) adenovirus infection of MCL cells despite the absence of receptor/coreceptor molecules known to be important for adenovirus infection of other cells types; 2) cytotoxicity of MCL cells after infection with specific adenovirus mutants; 3) a high degree of cytotoxicity after infection of some patient samples with viruses lacking the E1B 19k ‘‘antiapoptotic’’ gene; and 4) cytotoxicity after infection with viruses containing mutations in E1A pRb or p300 binding. The extent of cytotoxicity with the panel of viruses demonstrated interpatient variability, but 100% cytotoxicity, as determined by molecular analysis, was detected in some samples. These studies provide the foundation for: 1) the development of adenoviruses as cytotoxic agents for MCL and 2) analyses of key regulatory pathways operative in MCL cells. Ó 2005 International Society for Experimental Hematology. Published by Elsevier Inc.
Mantle cell lymphoma (MCL) is a distinct subtype of nonHodgkin’s lymphoma (NHL) derived from CD5D B cells originating from the mantle zone surrounding reactive germinal centers. MCL accounts for 5 to 8% of adult NHL in the United States and Europe. The disease is aggressive with a median patient survival of 2 to 4 years. Although standard chemotherapeutic regimens generally induce disease response, the responses are often partial and of short duration. Even when a complete remission is obtained, the median duration is short with no plateau in failure-free survival [1–7]. Improved outcomes occur with more intensive regimens with or without autologous or allogeneic hematopoietic stem cell rescue/transplantation; however, many patients still relapse and these intensive therapies are difficult to administer, are associated with many adverse effects, and are often limited to younger patients. Hence, MCL is an ideal disease for the development of novel therapeutic agents.
Offprint requests to: Daniel J. Medina, Ph.D., The Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901; E-mail: [email protected]
MCL is characterized by the proliferation of CD5D, CD19D, CD232 monoclonal B cells containing the chromosomal translocation t(11;14) (q13;q32), which places the cyclin D1 gene (BCL-1/CCND1/PRAD-1) under the transcriptional control of immunoglobulin heavy-chain enhancer elements [8–10]. The resulting overexpression of cyclin D1 is thought to play a key role in the pathogenesis of MCL. Cyclin D1 is important in regulating the G1-to-S phase transition of the cell cycle [11]. Cyclin D1 interacts with cyclin-dependent kinases (Cdk) 4 and 6 to form a complex that phosphorylates the retinoblastoma protein (pRb), causing the release of E2F transcription factors, which are essential for the expression of S-phase genes. Recent studies indicate that loss of function mutations/deletions in cell cycle regulatory proteins, ataxia telangiectasa mutated (ATM), and altered expression of apoptotic regulatory proteins expression also contribute to the development of MCL [10,12–16]. Therefore, multiple genetic events targeting cell cycle, response to DNA damage, and induction of apoptosis contribute to mantle cell lymphomagenesis. The genetic alterations that mediate cell cycle progression, repression of cellular response to DNA damage and inhibition of apoptosis in MCL cells, are analogous to
0301-472X/05 $–see front matter. Copyright Ó 2005 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 10.1016/j.exphem.2005.07.009
D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
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virus-induced changes that occur during adenovirus, human papillomavirus, or polyomavirus infection. For example, during a productive adenovirus infection, the virus induces cellular pathways that support cell cycle progression and DNA replication while suppressing DNA repair and apoptosis. This strategy allows cellular support of viral DNA synthesis while allowing the cell to survive long enough to produce progeny virus. To achieve cell cycle progression without induction of apoptosis, adenovirus gene products interact with many of the same strategic regulatory pathways that are altered in malignant cells such as MCL cells (Fig. 1). For example, adenovirus gene products target p53, pRb, p300, DNA damage pathways, and apoptotic pathways. Hence there may be redundant targeting of key pathways when an adenovirus infects a malignant cell [17–20]. As a result, attenuated/mutated adenoviruses may be constructed to infect and conditionally replicate or induce cytotoxicity in tumor cells, as previously demonstrated for malignant cells with p53 mutations [17,18]. In those studies, attenuated adenoviruses containing a mutation that prevented inactivation of p53 selectively killed p53-deficient epithelial cells. In this study,
we demonstrate adenovirus infection of primary MCL cells and use a panel of attenuated adenoviruses to infect and kill MCL cells. This study was prompted by the knowledge that multiple perturbations of several important cellular pathways in MCL may provide a cellular milieu that complements specific adenovirus mutations, allowing the development of mutant viruses with selective cytotoxicity. For example, adenovirus early gene products encoded by the E1 region (E1A and E1B) are expressed shortly after infection. E1A encodes two major overlapping polypeptides, a 286 amino acid polypeptide and a 243 amino acid polypeptide. The 286 polypeptide contains a functional domain, complementary region 3 (CR3), that regulates viral gene expression. In addition, both of the E1A polypeptides interact with a variety of host transcription and chromatin remodeling factors, including pRb and p300. The other E1 region, E1B, also encodes key regulatory proteins. The E1B 55-kd polypeptide interacts with p53 and the E1B 19-kb polypeptide inhibits apoptosis [17–20]. As these E1 gene products modulate key cellular pathways such as pRb and p53 that are altered in MCL cells, we hypothesized that specific viral
CYCLIN D1
S phase viral gene expression
E2F p300 pRb
E1A
Early gene expression CR 3 pRb
p300 binding
orf 4
E4 induction
apoptosis
orf 3/6 binding
p14 arf
E1B19K
antiapoptotic
E1B55K
p53
altered p53-mdm2 interaction
ATM cell cycle arrest
p53
bax, CD95
bcl-2 IAP apoptosis
cellular stress altered transcriptional co-activation
mdm2
Figure 1. A simplified model of adenovirus protein–MCL cell interactions. Adenovirus E1A and E1B are expressed early during infection. E1A complementary region (CR) 1 and 2 are included on both a large (286 aa) and small (243 aa) polypeptide. CR3 is unique to the large E1A polypeptide and activates adenovirus early gene expression. The E1A pRb and p300 binding sites are present in both polypeptides. E1A p300 binding may impact on cytotoxicity by modulating host and viral transcription, increasing p53 (impairment of mdm2 induction or alteration of mdm2-p53 interactions) and forming a complex with pRb. E1A pRb binding induces E2F family members (as well as other proteins), resulting in induction of several genes required for G1-S transition and adenovirus DNA synthesis. p14arf is also induced by E1A and may enhance p53 stability. Stabilized p53 may result in proapoptotic bax or CD95 expression. To counteract the proapoptotic effects of p53 expression, the E1B region encodes: 1) a 55K protein that binds p53 and collaborates with E4-encoded peptides (open reading frames [orf]3/6) to transport and degrade p53 and 2) a 19k protein that has potent antiapoptotic activity. Other early proteins encoded by E4 may modulate apoptosis by binding to cellular phosphatases and/or inhibiting DNA repair complexes induced by viral DNA. Cyclin D1 overexpression in MCL may result in many biological effects, including pRb hyperphosphorylation, which could complement E1A pRb binding.
D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
mutants, such as those that alter E1A ‘‘inactivation’’ of pRb, might be complemented by cyclin D1 overexpression and result in selective cytotoxicity. In this study we tested the above hypothesis and demonstrate that infection of MCL cells with a panel of mutated adenoviruses, including those with specific mutations in E1A, results in cytotoxicity. Furthermore, we demonstrate infection of MCL cells via a novel coxsackie-adenovirus receptor (CAR)-independent pathway not observed in previous studies with chronic lymphocytic leukemia (CLL) and multiple myeloma [19,20]. These studies provide preclinical information that will foster the development of attenuated adenoviruses as therapeutic agents for patients with MCL and also provide the foundation for future molecular analyses to define the dynamic activity of specific key regulatory pathways in MCL. Material and methods Patients Six MCL patients (samples MCL-1–MCL-6) were included in this study. Diagnosis was based on immunophenotype (CD5D, CD19D, and CD232) of malignant cells in conjunction with expression of cyclin D1 and/or detection of the translocation t(11;14) by cytogenetics or fluorescence in situ hybridization analysis. Samples of blood or spleen were obtained under a protocol approved by the Robert Wood Johnson Medical School Institutional Review Board. Mononuclear cells were isolated from 10 to 25 mL of fresh heparinized blood or splenectomy sample by Histopaque centrifugation. Isolated cells were counted, resuspended in freezing medium (90% fetal bovine serum and 10% dimethylsulfoxide), and stored in liquid nitrogen until use. Analyzed MCL cells obtained from blood were greater than 95% MCL cells as determined by flow cytometry (MCL-1, 97%; MCL-2, 95%; MCL-3, 97%; MCL-4, 98%; MCL-5, 96%; MCL-6, 97%). The sample obtained from the spleen of patient MCL-1 was 75% MCL cells, as determined by flow cytometry. Cells and viruses Two hundred ninety-three cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with antibiotics, 2 mM L-glutamine, and 10% fetal bovine serum (FBS). MCL cells were maintained ex vivo in RPMI 1640 medium supplemented with antibiotics, 2 mM L-glutamine, and 10% FBS. The viruses used in the experiments, Ad5dl309, E1A-, Pac3, and Ad5dl337, were described previously [19–22]. The wild-type parent for these mutant viruses, Ad5dl309, lacks a portion of the E3 region that has been shown to be dispensable for growth in cell culture [23]. The following E1A mutant adenoviruses were generously provided by Elizabeth Moran (Temple University, Philadelphia, PA, USA): 12S.RG2, E1A.RG2, 12Swt, 12S.YH47, and E1A.YH47 [24]. All viruses were grown and titered on 293 cells at 37 C. For mock infections, Ad5dl309 was heat-inactivated at 65 C for 30 minutes. The recombinant adenovirus containing the green fluorescent protein (GFP) was purchased from Quantum Biotechnologies (Montreal, Quebec, Canada). That virus (AdGFP) contains the GFP gene under the control of a cytomegalovirus (CMV) promoter, in the E1 region of adenovirus. A summary of the viruses is presented in Table 1.
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Table 1. Viruses used in this study
Virus Ad5 dl309 Ad5 E1A2 Pac3 Ad5 dl337 E1A 12S E1A YH47 928 E1A RG2 12S YH47 928 12S RG2
E1A p300
E1A pRb
E1A CR3
E1B 55K
E1B 19K
D 2 2 D D D 2 D 2
D 2 2 D D 2 D 2 D
D 2 2 D 2 D D 2 2
D D 2 D D D D D D
D D 2 2 D D D D D
Infection MCL cells (2 3 106) were infected with the indicated viruses at a multiplicity of infection (moi) of 100 for 4 to 6 hours at 37 C. After infection, the cells were washed three times with Dulbecco’s phosphate-buffered saline to remove nonadsorbed virus. The cells were then pelleted by centrifugation at 500g for 10 minutes, resuspended in 2 mL of medium, and culture in 24-well plates. At the indicated times, samples were removed for determination of cell viability by trypan blue staining and virus yield. Heat-inactivated Ad5dl309 were used for mock infections. Virus assay Cell-free supernatants were collected at the indicated times to assess virus production. The virus titer was determined by plaque assay on 293 cells. Briefly, 1 mL of serial 10-fold dilutions of cellfree supernatant were added to duplicate 60-mm dishes containing a 95% confluent layer of 293 cells for 1 hour at 37 C. After incubation, the supernatant was removed and the plates were overlaid with agarose as described [25]. Viral plaques were counted on day 14 and the viral titer was determined Flow cytometric analysis for CAR and integrin expression Cell surface expression of CAR was determined by indirect immunofluorescence flow cytometry. The primary monoclonal antibody (mAb) RcmB anti-CAR (provided by Robert Finberg Dana-Farber Cancer Institute, Boston, MA, USA) was used at a 1:100 dilution and visualized using fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (Sigma, St. Louis, MO, USA) secondary antibody at 1:200 dilution. Cell surface expression of integrins was determined by flow cytometry using FITC-conjugated antibodies (1 mg/mL) for avb3, avb5, b1, and aM (Chemicon, Temecula, CA, USA). In every experiment an irrelevant isotype-controlled mAb was used to determine background staining. All staining procedures were for 30 minutes at 4 C in Hanks balanced salt solution (HBSS) that contained 5% FBS. Flow cytometric analysis was performed using a FACScan analyzer. Immunoglobulin heavy-chain analysis To assess molecular responses after adenovirus infection of MCL cells, primary cells from the spleen of patient MCL-1 were infected with Ad5dl309 at an moi of 100 and washed three times with PBS. Mock (heat-inactivated Ad5dl309) infection of MCL cells was used as a control. At the indicated time, 2 3 104 cells from each of the infections was mixed with 3 3 106 uninfected Jurkat T cells (as a carrier). Viable cells were obtained by Ficoll-hypaque density separation and the isolated DNA was
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polymerase chain reaction (PCR) amplified with allele-specific primers for patient MCL-1 [26]. Additional controls included DNA isolated from Jurkat cells, uninfected MCL-1 cells, and cells from another patient with MCL. This last control was used to demonstrate the specificity of the allele-specific primers. Statistics The mock infection was treated as the control group. Therefore, each specific virus was analyzed independently at each time point versus the mock infection using Student’s t-tests, at the 0.05 level of significance. Further analyses were completed using a one-way analysis of variance, at the 0.05 level of significance, to determine the statistical differences between baseline and all subsequent time points postinfection for each of the viruses and the mock infection. Analyses were completed using Microsoft Excel 2000 (Redmond, WA, USA) and SPSS Version 11.5 (Chicago, IL, USA).
Results Infection of primary MCL cells with AdGFP Initial experiments established the capacity of adenoviruses to infect MCL cells. In these studies, a recombinant
MCL-1
adenovirus expressing GFP under the control of a CMV promoter (AdGFP) was used to infect MCL cells. Five days after infection, the number of cells expressing green fluorescence was measured by flow cytometry. In each MCL sample a marked shift in fluorescence was observed after infection with AdGFP (Fig. 2). In all cases, the mean intensity of the entire population shifted in a statistically significant fashion, indicating infection of a large percentage of cells. In contrast, as previously described, neither normal B cells obtained from tonsil infected with AdGFP [19] nor MCL cells infected with a control virus not containing the GFP gene (Fig. 2) demonstrated any statistically significant shift in intensity after infection. Cell surface expression of CAR and integrins on MCL cells In permissive cells, adenovirus infection is mediated by the quantity of both CAR and av integrins expressed on the surface of the target cells [27–30]. Lymphocytes, however, are generally resistant to adenovirus infection, in part because they normally lack expression of CAR and/or av integrins [31–34]. Because MCL cells from different patients showed variable infection efficiency with AdGFP, we
MCL-2
MFI P=0.01
MCL-4 MFI P=0.04
MCL-6
MCL-4 (Pac3)
MCL-5 MFI P=0.02
MFI P=0.02
MFI P=0.01
MFI P=0.29
CLL
Figure 2. MCL cells can be infected with an adenovirus encoding GFP. MCL cells were infected with AdGFP (moi 5 100), containing the GFP gene under control of a CMV promoter. Five days after infection, cells were assayed for GFP expression by flow cytometry. The bold line represents the fluorescence of AdGFP-infected cells. The lighter line histogram represents the fluorescence of uninfected cells. The panel labeled MCL-4 (Pac3) represents a control infection of MCL cells with an adenovirus not expressing GFP. The panel labeled CLL represents AdGFP infection of CLL cells and was used as a positive control, as described by Medina et al. [19]. Statistical analysis of the shift in mean fluorescence intensity after infection is indicated in the upper right corner of each panel.
D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
characterized the expression of CAR and integrins avbb3, avb5, b1, and aM on MCL cells from five patients, as well as normal tonsil derived B cells by flow cytometry. The results are summarized in Table 2 and a representative histogram (MCL-4) is shown in Figure 3. As shown in Table 2, tonsil-derived B cells as well as cells MCL-1, MCL-2, MCL-4, and MCL-5 did not express CAR or avb3, avb5, and aM. MCL-6 expressed only low levels of CAR and was also negative for avb3, avb5, and aM expression. Only b1 integrin was consistently expressed at high levels in both tonsil-derived B cells and MCL cells. In contrast, high-level CAR expression has been detected on CLL cells [20,34]. These data suggest that alternate receptors may mediate adenovirus infection of MCL cells. Replication-competent adenovirus (Ad5dl309) is cytotoxic to MCL cells To determine whether adenoviruses induce cytotoxicity after infection of MCL cells, MCL-1 cells were infected with the parental virus Ad5dl309 or Ad5dl309 mutants containing deletions of either E1A (E1A2) or deletions of E1A and E1B (Pac3; see Table 1). This population of MCL cells was prepared from spleen and contained w75 % MCL cells. Cell viability was monitored over the course of 10 days by trypan blue exclusion. After 10 days in culture, heat-inactivated Ad5dl309-infected cells (mock-infected control), E1A2 virus-infected cells, and Pac3-infected cells had no loss of cell viability. In contrast, cytotoxicity was detected after Ad5dl309 infection, with viability reduced by 80% compared to control cells (Fig. 4). To determine whether residual viable MCL cells remained after 10 days of Ad5dl309 infection of MCL-1 cells, cellular DNA from viable mock-infected and Ad5dl309-infected cells were assayed for allele-specific immunoglobulin H (IgH) rearrangement by PCR using patient-specific primers. Mock-infected MCL-1 cells remained positive for the allelespecific IgH rearrangement while MCL-1 cells infected with Ad5dl309 demonstrated complete eradication of a PCR signal corresponding to the allele-specific IgH rearrangement (Fig. 5). Although only a qualitative PCR assay was performed, limiting dilution experiments (not shown) indicated
Table 2. CAR and integrin expression on MCL cells and tonsil B cells* Patient
CAR
b1
aM
aVb3
aVb5
Tonsil-1 MCL-1 MCL-2 MCL-3 MCL-4 MCL-5 MCL-6
4.6 0.1 2.8 ND 5.5 0.6 15
34.2 50.1 79.6 ND 10.8 83.1 97.2
6.5 1.4 1.0 ND 1.6 1.8 1.8
8.2 4.3 5.2 ND 10.4 5.1 5.1
2.3 1.2 3.3 ND 2.2 1.3 5.3
*Expressed as the % of cells staining positive with the indicated FITClabeled antibody compared to FITC-labeled isotype control.
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that Ad5dl309 infection resulted in at least a 3 log reduction in MCL cells. Mutated adenoviruses are cytotoxic to MCL cells Previous studies from our laboratory examining adenovirus-mediated cytotoxicity after infection of primary CLL cells demonstrated high patient variability with regards to cytotoxic effects induced by a given virus [19]. Therefore, we examined the use of attenuated adenoviruses in five additional MCL patients. In this experiment, MCL cells were infected with Ad5dl309, E1A2, Pac3, or an Ad5dl309 mutant containing a deletion in the E1B 19k gene (Ad5dl337; see Table 1). The cells were infected as described above and the cytopathic effects of the viruses were evaluated at the indicated time points by trypan blue exclusion. Nine to 12 days after infection with Ad5dl309, MCL-2, MCL3, MCL-4, MCL-5, and MCL-6 cells exhibited 65 to 80% cytotoxicity (Fig. 6). Infection with adenovirus deleted in both E1A and E1B (Pac3) resulted in minimal cytotoxicity (10–30%) after infection of MCL-2, MCL-4, MCL-5, and MCL-6. However, MCL-3 cells demonstrated O50% cytotoxicity on day 9. E1B 19K2 (Ad5dl337) resulted in marked cytotoxicity after infection of most of the samples, with near complete cytotoxicity of MCL-2 cells by day 12. MCL cells infected with the E1A2 adenovirus also demonstrated variable cytotoxicity. MCL-2 and MCL-3 demonstrated 75% cytotoxicity after infection with the E1A2 virus, whereas MCL-1, MCL-4, and MCL-6 demonstrated lesser degrees of cytotoxicity after infection with E1A2. Infection of MCL cells with adenoviruses containing E1A p300-binding and E1A pRb–binding mutations As a result of the t(11;14) translocation, MCL cells overexpress cyclin D1, which may result in phosphorylation of pRb and complement adenovirus E1A pRb binding. To begin to assess the ability of cyclin D1 to complement E1A activity, viruses with specific mutations in E1A were studied. These viruses had mutations in E1A p300–binding (RG2) or pRb-binding (YH47) sites in the context of either full E1A (13S) or a CR3-deleted E1A (12S; see Table 1). Adenoviruses containing the full E1A gene (13S) were cytotoxic, and cytotoxicity was maintained by individual mutations of either the E1A p300– or pRb-binding site (Fig. 7). However, in all patient samples for which there were enough cells to perform three independent infections with each virus (i.e., MCL-2, MCL-3, MCL-4, MCL-6), E1A.RG2 (with the p300-binding mutation) demonstrated statistically greater cytotoxicity ( p ! 0.05) than E1A.YH47 (with the pRb-binding mutation). When studied in the context of viruses containing deletion of E1A CR3 (12S.RG2 versus 12S.YH47, see Table 1), three of the four samples (MCL-3, MCL-4, MCL-6) had statistically greater cytotoxicity ( p ! 0.05) after infection with the virus containing p300-binding mutation. In the fourth sample (MCL2), the reverse pattern was seen after infection with
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D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
Figure 3. Cell surface expression of CAR and integrins on MCL cells. MCL cells were labeled with the respective monoclonal antibodies and analyzed by flow cytometry. The open histogram represents staining after reaction with an isotype control antibody. The shaded histogram represents staining with antibodies to adenovirus receptor CAR and the integrins b1, aM, avb3, and avb5.
12S.RG2 and 12S.YH47. The discrepancy in cytotoxicity after infection of MCL-2 (enhanced killing with viruses containing p300 mutation in the context of a full-length virus but decreased killing with viruses containing p300 mutation in the context of the 12S virus) may reflect the genetic heterogeneity of MCL and the complexity of virus–host interactions in the setting of these interpatient genetic differences. These data suggest that in several patient samples and virus backgrounds viruses that contain mutations that inhibit E1A-pRb binding are not fully complemented by cyclin D1 overexpression in MCL cells.
Discussion Previous work by others has established the capacity of adenovirus mutants to selectively infect and kill malignant epithelial cells containing p53 mutations [17,18]. The intent of this study was to determine whether adenoviruses with specific cytotoxicity for MCL cells could be developed. In our initial studies, MCL and CLL cells, but not tonsilderived B cells, were infected with adenovirus. MCL cells from different patients demonstrated variable infection efficiency. To determine whether some of this variation was due to different expression levels of cell surface molecules
D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
MCL-1 140
% of Control
120 100 80 60 40
*
20 0
Mock
dl309
E1A
Pac3
Figure 4. Adenovirus infection of MCL cells induces cytotoxicity. Cells from the spleen of patient MCL-1 were infected with Ad5dl309, mock-infected (heat-inactivated Ad5dl309), an E1A-deleted adenovirus (E1A2), or an adenovirus with a deletion of E1A and E1B (Pac3). At the indicated time cell viability was determined by trypan blue staining and compared to the viability of uninfected control cells. The asterisk indicates a statistically significant ( p ! 0.05) difference in cytotoxicity after infection compared to mock-infected control.
known to function as adenovirus receptors and coreceptors [27–30], we evaluated the expression of CAR and the coreceptors av integrins on MCL cells. Normal B cells were not efficiently infected by adenoviruses and had no/low expression of both CAR and avb3, avb5, and aM integrins [19,20]. In contrast, CLL cells, which can be infected by adenoviruses, uniformly expressed the CAR receptor and avb3, avb5 integrins but not aM integrins [20]. Surprisingly, MCL cells were infected at a level comparable to that seen for CLL cells but expressed undetectable or minimally detectable levels of CAR and coreceptors avb3, avb5, and aM integrins. These data suggest that adenovirus entry into
std
1
2
3
4
5
6
Figure 5. Molecular (PCR) analysis of adenovirus-mediated cytotoxicity of MCL cells. MCL cells from patient 1 were infected with Ad5dl309 or mock-infected with heat-inactivated Ad5dl309. The cells were maintained for 10 days and the surviving cells were assayed by qualitative IgH allele-specific PCR. Lanes: std, marker; 1, H2O; 2, Jurkat cell DNA; 3, DNA from MCL-1 infected with Ad5dl309; 4, DNA from MCL-1 mock-infected; 5, DNA from patient MCL-2; 6, DNA from uninfected MCL-1 cells.
1343
MCL cells is mediated by pathways distinct from those operative in CLL cells and independent of CAR and/or avb3, avb3, and aM integrins. Therefore, CAR expression cannot be used as a surrogate for determining the capacity of the cell to be infected by adenoviruses and novel adenovirus ‘‘receptors’’ may be present on MCL cells. Studies are currently underway to elucidate the mechanisms associated with adenovirus binding/internalization in MCL cells. In addition, because CLL cells, but not MCL cells, express high levels of CAR we are evaluating the absence of CAR expression as a distinguishing immunophenotypic characteristic of MCL (in comparison to other lymphoproliferative disorders). Having established that adenoviruses infect primary MCL cells, infection with a panel of adenovirus mutants was undertaken to determine their spectrum of cytotoxicity in individual patient samples. These studies demonstrated that attenuated adenoviruses are capable of inducing cytotoxicity in MCL cells and that the degree of cytotoxicity induced by individual viruses varied among MCL samples. This patient variation in adenovirus-induced cytotoxicity with individual viruses is similar to that reported for adenovirus infection of CLL cells [19] and is most likely the result of genetic variation in the individual patient malignant cells. Of the viruses examined in the first set of infections, the replication-competent Ad5dl309 and the mutated Ad5dl337 (E1B 19K deletion) had the broadest range and extent of cytotoxicity. The most attenuated virus, Pac3 (deletion of E1A and E1B), induced the least toxicity in most samples. The adenovirus deleted in E1A (E1A2) demonstrated variability in terms of cytotoxicity. Hence, E1A activity is not uniformly and completely complemented by cyclin D1 overexpression in MCL cells. This lack of full E1A complementation by cyclin D1 overexpression likely reflects the multiple functions and binding partners of E1A during the viral life cycle [35–39]. To refine the studies of adenovirus E1A mutant viruses, attenuated viruses with mutations in either pRb or p300 binding were studied. As cyclin D1 binds and phosphorylates pRb, facilitating E2F-1 release and cell cycle progression, it was hypothesized that an attenuated adenovirus containing a mutation in E1A pRb binding might be fully complemented in MCL cells. Although cytotoxicity was seen after infection with this E1A pRb binding mutant virus, the extent of cell killing was reduced in comparison to infection with a virus containing intact E1A or, in several patient samples, a virus with an E1A mutation in p300 binding. Therefore, E1A pRb binding is not fully complemented by overexpression of cyclin D1. The relative lack of E1A-pRb-binding complementation in MCL cells may relate to a lack of complete pRb phosphorylation by cyclin D1 or to more complex interactions between cyclin D1, E1A, and pRb. For example, hyperphosphorylation of pRb and increased E2F-1 expression has been detected in a subset of patients with MCL, but
D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
1344
MCL-2 120
*
*
*
*
80 60 40
100
20
80
* *
40
0
4
8
3
12
Mock dl309 E1APac3 dl337
120
% of Control
* *
*
60
*
*
*
40
0
3
100 80 60 40
0
9
6
3
Days
MCL-6
*
*
6
9
Days
120
% of Control
Mock dl309 E1APac3 dl337
20
20
100
9
MCL-5
120
% of Control
MCL-4
*
6
Days
Days
80
*
*
60
20
0
100
Mock dl309 E1APac3 dl337
120
% of Control
% of Control
100
MCL-3
Mock dl309 E1APac3 dl337
*
Mock dl309 E1APac3 dl337
*
80
*
*
60 40 20 0 3
6
9
Days Figure 6. Attenuated adenovirus infection of MCL cells induces cytotoxicity. Primary MCL cells from five patients were infected with Ad5dl309, heat-inactivated Ad5dl309 (Mock), an E1A-deleted adenovirus (E1A2), an adenovirus with a deletion of E1A and E1B (Pac3), or an adenovirus containing an E1B 19K deletion (Ad5dl337). At the indicated times, cell viability was determined by trypan blue staining and compared with the viability of uninfected cells. Asterisks indicate statistically significant differences in cytotoxicity ( p ! 0.05) compared to mock-infected controls.
there is no clear correlation of pRb phosphorylation with the degree of cyclin D1 overexpression [15,16]. In fact, there are many other biological activities of cyclin D1 that might contribute to oncogenesis. For example, pRb interaction without phosphorylation, cyclin D1-induced transcriptional modulation, cyclin D1 sequestration of cyclin-dependent kinases and inhibitors, and/or cyclin D1-mediated antiapoptotic effects have been described [40–45]. Transcriptional modulation via interaction with (and sequestration of) the transcription factor DMP1 may be relevant, as DMP1 is required for the induction of p14arf, a regulator of p53 induction by a variety of oncogenic stimuli, including adenovirus early polypeptides
[45–48]. Hence, there are many potential mechanisms by which cyclin D1 overexpression might contribute to MCL lymphomagenesis in the absence of direct effects on pRb. In fact, overexpression of cyclin D1 and p16INK4A loss can be seen concurrently in MCL, suggesting that there may be additional independent, nonoverlapping mechanisms by which both pRb inactivation and cyclin D1 contribute to MCL generation/maintenance [49]. In addition, mutational analysis of cyclin D1 has demonstrated that the pRb kinase domain is dispensable for cellular transformation in some assays [50]. Hence, cyclin D1 overexpression may mediate oncogenic effects other than pRb phosphorylation.
D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
% of Control
120 100
*
80
*
60
*
40
MCL-3 120 100
% of Control
Mock dl309 12S.RG2 E1A.RG2 12S wt 12S.YH47 E1A.YH47
MCL-2
*
80
4
40
8
3
12
Days Mock dl309 12S.RG2 E1A.RG2 12S wt 12S.YH47 E1A.YH47
% of Control
100
*
* *
40
MCL-5 120 100
% of Control
MCL-4
60
6
10
Days
120
Mock dl309 12S.RG2 E1A.RG2 12S wt 12S.YH47 E1A.YH47
80 60 40 20
20 0
0 3
6
9
MCL-6
120
*
80
3
6
9
Days
Days
% of Control
*
60
0
0
100
*
Mock dl309 12S.RG2 E1A.RG2 12S wt 12S.YH47 E1A.YH47
20
20
80
1345
Mock dl309 12S.RG2 E1A.RG2 12S wt 12S.YH47 E1A.YH47
* *
60 40 20 0 3
6
9
Days Figure 7. Infection of MCL cells with adenoviruses containing E1A-pRb- or E1A-p300-binding mutants induce cytotoxicity. Primary MCL cells from five patients were infected with the indicated viruses at an moi of 100. At the indicated times, cell viability was determined by trypan blue staining and compared to mock-infected cells. The parental virus, Ad5dl309, contains the complete E1A region; E1A.RG2 has a mutation of the E1A-p300 binding site; E1A.YH47 has a mutation of the E1A-pRb binding site. The 12Swt lacks the CR3 but retains the E1A-p300 and -pRb binding sites; 12S.RG2 lacks E1A CR3 and has a mutation of the E1A-p300 binding site; 12S.YH47 lacks E1A CR3 and has a mutation of the E1A-pRb binding site. Asterisks indicate infections that result in statistically significant greater cytotoxicity ( p ! 0.05) than the control (mock-infected) cells. Statistical comparisons of E1A.RG2 and E1A.YH47 infection and 12S.RG2 and 12S.YH47 infections are described in the text.
Much of what is known concerning cyclin D1 and other regulatory proteins involved in the control of cell cycle, apoptosis, stress response, and transcription is based upon molecular analysis of cell lines and transgenic mice. Such studies have provided fundamental information concerning key regulatory interactions, but variable biological effects are often seen in different cell types and contexts. This dependence upon cell type and physiology emphasizes the importance of analyzing basic regulatory protein interactions in primary malignant cells. Studies of adenovirus infection in these cells may allow a dynamic analysis of
regulatory pathways active during infection with selected adenovirus mutants and may identify key regulatory pathways that are active in individual patient MCL cells. The variability in outcomes after infection with different viruses implies an interpatient heterogeneity in the activity of regulatory and/or oncogenic pathways. Studies such as these will hopefully allow these pathways to be better characterized in primary cells and, ultimately, targeted by either attenuated adenoviruses or other directed therapies. In summary, this report demonstrates MCL cell infection with adenoviruses despite absent or very low-level
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D.J. Medina et al./ Experimental Hematology 33 (2005) 1337–1347
expression of CAR or selected integrins known to mediate adenovirus infection in other cells. Adenovirus-mediated cytotoxicity is demonstrated after infection of MCL cells, and there is variable cytotoxicity after infection with viruses attenuated by specific mutation of adenovirus E1 genes. The requirement of E1A-pRb binding for maximum cytotoxicity implies that cyclin D1 expression cannot completely complement E1A-pRb binding under the conditions of the infection. In addition, adenovirus-mediated cytotoxicity after infection of MCL cells with attenuated viruses that do not result in cytotoxicity after infection of normal B cells raises the prospect of developing attenuated adenoviruses for therapeutic use in patients with MCL.
Acknowledgments Supported by Lymphoma Research Foundation and the Virginia L. Koehler Lymphoma Grant Program, The New Jersey Commission on Cancer Research.
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