Inhibition of Mitochondrial Matrix Chaperones and Antiapoptotic Bcl-2 Family Proteins Empower Antitumor Therapeutic Responses Free

Mitochondrial Hsp-90 (mtHsp90) has been shown to be of utmost importance for cancer cell survival and growth (1). Gamitrinib-triphenylphosphonium (G-TPP) is a synthetic small-molecule Hsp90 ATPase antagonist with preferential tropism to mitochondria (2). In preclinical studies, G-TPP was shown to have inhibitory effects on various proneoplastic features in different cancer types (2–8).

The antiapoptotic Bcl-2 family members regulate cell death at the outer mitochondrial membrane (9–14). Because Bcl-2 and Bcl-xL are frequently increased in cancer cells and bear a major inhibitory impact on apoptosis, a novel class of proapoptotic compounds, called BH3 mimetics, such as ABT263 or ABT199 (15, 16), was developed. However, they fail to inhibit Mcl-1. Therefore, strategies need to be tailored that lower Mcl-1 levels in tumor cells.

In this report, we demonstrate that gamitrinib decreases protein levels of both Mcl-1 and its deubiquitinase Usp9X by activation of the integrated stress response. Consequently, we tested the hypothesis that interference with mitochondrial matrix chaperone proteins combined with the inhibition of antiapoptotic Bcl-2 family members would facilitate cancer cell death as a consequence of a proapoptotic mitochondria-specific “dual-hit.” This hypothesis was supported by an earlier drug screen that demonstrated that BH3 mimetics and gamitrinib are potentially synergistic (7). Our data show that disruption of the Hsp90 chaperone network when combined with BH3 mimetics yields a synergistic antiproliferative and proapoptotic effect across a wide panel of different cancer cells. Moreover, combined treatment with the mitochondrial chaperone inhibitor G-TPP and BH3 mimetics results in a significant enhancement of tumor growth inhibition in several in vivo model systems, including patient-derived xenografts.

ABT263, GX15-070, ABT199, WEHI-539, and A-1210477 were purchased from Selleckchem. G-TPP was synthesized as described previously (4).

U87MG, LN229, U251, and T98G human glioblastoma cell lines and Colo-829 and MeWo were obtained from the ATCC. WC62 melanoma cells were from Coriell Cell Repositories. NCH644 and NCH421K stem cell–like glioma cells were obtained from Cell Line Services (CLS). The respective cell line depository authenticated the cells. U87-EGFRvIII cells were kindly provided by Dr. Frank Furnari (Ludwig Institute for Cancer Research, La Jolla, CA). The GS9-6 (17) are primary neurosphere stem-like glioma cells derived at the University of Massachusetts (Worcester, MA). All cell lines were obtained between 2013 and 2015. The MGPP-3 (p53^−/−, PTEN^−/−, PDGFR⁺) are murine proneural glioblastoma cells. All cells were cultured as previously described (12, 18–21).

To examine cellular proliferation, MTT assays were performed as described previously (18, 22, 23).

For Annexin V/propidium iodide (PI) staining, the Annexin V Apoptosis Detection Kit (BD Pharmingen) was used as described previously (24, 25). For PI staining, cells were resuspended in 300 μL PBS and fixated by adding 1,000 μL ice-cold ethanol prior to incubation overnight at 4°C. Then the cells were centrifuged at 1,800 rpm, the supernatant was removed and 400 μL PI/RNase staining solution (Cell Signaling Technology) were added prior to incubation for 15 minutes at room temperature and flow cytometric analysis. To detect intrinsic apoptosis staining and loss of mitochondrial membrane potential, tetramethylrhodamine, ethyl ester staining was performed according to the manufacturer's instructions (Mitochondrial Membrane Potential Kit, Cell Signaling Technology). The data were analyzed with the FlowJo software (version 8.7.1; Tree Star).

Briefly, cells were incubated for 6 hours with the formed complexes of Oligofectamine 2000 (Invitrogen) and the respective siRNA (12-well condition) in DMEM without FBS and antibiotics. After 6 hours, FBS was added to a total concentration of 1.5%.

Specific protein expression in cell lines was determined by Western blot analysis as described before (26).

Patient-derived xenograft cells were stereotactically injected into 6- to 8-week-old male or female nude GFP or SCID SHO mice as previously described (22, 24). A burr hole was positioned 2 mm anterior and 2 mm lateral of the bregma prior to introducing a Hamilton syringe under stereotactic guidance 3 mm into the striatum. Motorized injections of the cells were performed at a rate of 0.5 μL/minute. MRI imaging was performed using a Bruker BioSpec, 9.4 Tesla imaging device. Survival was assessed by calculating Kaplan–Meier curves.

A total of 1 × 10⁶ U251, LN229, U87MG, A375, or A375R cells were implanted subcutaneously into the flanks of 6- to 8-week-old SCID SHO mice as described before (22, 24). Measurements were performed with a caliper and tumor sizes were calculated as (length × width²)/2. Treatment was performed intraperitoneally twice a week for 3 weeks. For intraperitoneal application G-TPP, ABT263 and GX15-070 were dissolved in 80% Cremophor EL (Sigma) and 20% Ethanol (Pharmco-Aaper; v/v).

Statistical significance was assessed by Student t test using Prism version 7.00 (GraphPad). A P ≤ 0.05 was considered statistically significant. Bliss analysis was performed to detect synergistic, additive, or antagonistic effects as described previously (20).

All procedures were in accordance with Animal Welfare Regulations and approved by the Institutional Animal Care and Use Committee at the Columbia University Medical Center.

To assess whether inhibition of mitochondrial Hsp90 chaperones primes tumor cells to apoptosis, a broad range of tumor cells were analyzed by MTT assay for viability after treatment with ABT263, GX15-070, G-TPP, or the indicated combination treatments (Fig. 1A). In U87MG, LN229, U251, and T98G established glioblastoma cells as well as in GS9-6 and NCH644 stem cell-like glioma cells, GBM6, GBM39 patient-derived xenograft glioblastoma cells, and in primary proneural murine glioblastoma cells (MGPP-3, derived from a transgenic model), a synergistic reduction in cellular viability was found when cells were treated with the combination of ABT263 and G-TPP (Fig. 1B and C; Supplementary Figs. S1 and S2; Supplementary Tables S1 and S2). In contrast, U87-EGFRvIII cells treated with the combination therapy of ABT263 and G-TPP showed only an additive effect (Fig. 1B; Supplementary Table S1). Next, we assessed whether these observations are restricted to glioblastoma cells. Similarly, BRAF-mutated melanoma cells, A375, BRAF-inhibitor resistant, A375R, WC62, MeWo, and COLO 829 melanoma cells as well as triple receptor-negative breast cancer cells, MDA-MB-468, displayed a synergistic reduction in viability after treatment with ABT263 and G-TPP (Fig. 1B; Supplementary Fig. S3; Supplementary Tables S1 and S3). Although the combination treatment did not yield a synergistic antiproliferative effect on pancreatic cancer cells, PANC1, the combination treatment revealed at least enhanced antiproliferative activity when compared with treatment with each compound alone (Supplementary Fig. S3; Supplementary Table S1). Akin to ABT263, GX15-070 broadly enhanced apoptosis and loss of cellular viability induced by mitochondrial matrix inhibitors in a synergistic manner (Fig. 1B and D; Supplementary Figs. S1 and S7; Supplementary Table S1). Given that ABT263 and GX15-070 are broad BH3 mimetics, we assessed as to whether selective BH3 mimetics would synergize with G-TPP as well. To this end, U87MG and T98G cells were treated with the selective Bcl-xL inhibitor WEHI-539, the selective Bcl-2 inhibitor ABT199 and the selective Mcl-1 inhibitor A-1210477 in the presence or absence of G-TPP. Our data show that G-TPP combined with each of these BH3 mimetics caused a synergistic reduction in cellular viability (Fig. 1E and F; Supplementary Table S4). Similar results were achieved in LN229 and GBM6 cells (Supplementary Table S4), suggesting that G-TPP primes mitochondria for broad and selective BH3 mimetics and that likely this involves in part the mitochondriotoxic properties of G-TPP. Combined treatment with ABT263 and G-TPP displayed typical features of apoptosis, such as enhanced cellular fragmentation and blebbing (Fig. 1G). In U87MG, U87-EGFRvIII, T98G, and LN229 glioblastoma cell lines enhanced DNA fragmentation was detected in cells treated with the combination of ABT263 and G-TPP (Supplementary Fig. S4A). U87-EGFRvIII cells were least responsive. It should be noted that strongest synergy in cell proliferation assays correlated with the most prominent DNA fragmentation/apoptosis. To further verify whether apoptosis induction is a relevant part of the mechanism, we performed Annexin V/PI staining in U87MG and T98G cells (Fig. 2A). Combined treatment with ABT263 and G-TPP resulted in a significant induction of apoptotic cell death as compared with either treatment alone. Consistently, both U87MG and T98G cells treated with ABT263/G-TPP displayed enhanced reduction of the mitochondrial membrane potential when compared with cells treated with each compound alone (Fig. 2B). T98G and LN229 cells exposed to the combination treatment revealed enhanced cleavage of both caspase-9 and caspase-3 (Fig. 2C). Furthermore, pan-caspase inhibition abolished the pro-apoptotic response following a combined treatment with ABT263 and G-TPP (Fig. 2D). In contrast, treatment with necrostatin, a necroptosis inhibitor, did not affect apoptosis driven by the combination therapy, which supports a caspase-mediated specific apoptotic response as mechanism (Fig. 2E). Next, we assessed to what extent selective BH3 mimetics induced apoptosis and mediated loss of the mitochondrial membrane potential in the presence or absence of G-TPP. U251, T98G, LN229, and U87MG established glioblastoma cells, NCH644 glioma stem cell-like cells, and MeWo and WC62 melanoma cells were treated with the selective BH3 mimetics, WEHI-539, ABT199, and A-1210477 alone and in combination with G-TPP. We found that G-TPP enhanced apoptosis for all selective BH3 mimetics (Fig. 2F and G; Supplementary Figs. S4B and S5). Similar findings were made with regards to loss of the mitochondrial membrane potential in WC62, MeWo, and T98G cells (Supplementary Fig. S6). To confirm the above findings with selective BH3 mimetics, we silenced the expression of Bcl-xL, Bcl-2, or Mcl-1 in LN229 glioblastoma cells (Fig. 3A and B). Our data show that cells silenced for Bcl-xL, Bcl-2, or Mcl-1 combined with G-TPP treatment displayed a significantly enhanced fraction of sub-G₁ cells, which recapitulates our findings with the selective BH3 mimetics we reported above (Fig. 3A). Concerning the relative contribution of each antiapoptotic Bcl-2 family member, it appears that this is cell type dependent.

To account for the role of Bax and Bak in ABT263/G-TPP-mediated cell death, we performed double-knockdown experiments for Bax and Bak. As shown in Fig. 3C, Bax/Bak–silenced LN229 cells treated with ABT263/G-TPP showed a marked reduction in the fraction of Annexin V-positive cells when compared with cells treated with n.t.-siRNA instead. For single-agent treatments, only minor effects were noted in the presence of Bax/Bak knockdown when compared with treatment with n.t.-siRNA. In line with this finding, dual Bax/Bak knockdown resulted in reduced cleavage of PARP and caspase-3 in cells treated with ABT263/G-TPP (Fig. 3D). For further clarification of the role of Bax in this setting, we conducted an immunoprecipitation for active Bax and found that ABT263, G-TPP, and the combination of both resulted in an activation of Bax, suggesting that Bax might be required for cell death execution, but that it is not solely responsible for it (Fig. 3E). To demonstrate that proapoptotic molecules are released upon treatment with the drug combination of ABT263 and G-TPP, LN229 cells were treated with ABT263, G-TPP, or the combination of both for 7 hours. Lysates were prepared and immunoprecipitated with an antibody against Mcl-1 (Fig. 3G and H). Immunoblots for BIM protein indicate that Mcl-1 avidly binds BIM protein in untreated LN229 cells. Notably, LN229 cells that were treated with ABT263 revealed increased binding of BIM to Mcl-1, suggesting that Mcl-1 counteracts the proapoptotic effect of Bcl-2/Bcl-xL inhibition by absorbing BIM protein. The combination treatment of ABT263 and G-TPP resulted in a release of BIM from Mcl-1, thereby facilitating intrinsic apoptosis. Moreover, specific silencing of BIM by siRNA attenuates caspase-3 cleavage induced by the combination treatment of ABT263 and G-TPP, further supporting the role of BIM in ABT263/G-TPP–mediated cell death (Fig. 3F).

Expression of Mcl-1 is a mechanism of resistance towards ABT199, ABT263, and ABT737. We therefore focused our next studies on determining effects of G-TPP on expression of Mcl-1. As shown in Fig. 4A, protein levels of Mcl-1 were decreased in LN229, U251, A375, and HL-60 cells following treatment with G-TPP in a dose-dependent manner. Consistent with this finding, expression of the deubiquitinating enzyme Usp9X was also markedly decreased. Notably, real-time RT-PCR analyses suggest that downregulation of both Mcl-1 and Usp9X is not mediated on the transcriptional level (Supplementary Fig. S8A and S8B). In contrast, both protein and mRNA levels of Noxa, a known proapoptotic interactor of Mcl-1, were consistently increased (Supplementary Fig. S8C). Given our observation that Mcl-1 and Usp9X are suppressed by G-TPP, we determined as to whether Mcl-1 or Usp9X levels determine the sensitivity of cancer cells toward the BH3 mimetic, ABT263. To this end, we knocked down Mcl-1 (Fig. 4B and C; Supplementary Fig. S9A–S9D) and Usp9X (Fig. 4D and E; Supplementary Fig. S9E–S9H) by siRNA and observed that both Mcl-1 and Usp9X knockdown potently sensitized LN229 and U251 glioblastoma cells and A375 melanoma cells to the cytotoxic effects of ABT263, indicating that Mcl-1 and Usp9X levels are key regulators in ABT263-mediated apoptosis. Next, we assessed whether the G-TPP–mediated reduction in Mcl-1 and Usp9X protein is posttranslationally driven. LN229 cells were treated with the protein synthesis inhibitor cycloheximide in the presence or absence of G-TPP. The protein stability of both Mcl-1 and its interacting deubiquitinase Usp9X was significantly decreased in the presence of a mitochondrial matrix inhibitor. This suggests that G-TPP suppresses Mcl-1 and Usp9X levels in a posttranslational manner (Fig. 4F). Furthermore, inhibition of proteasomal degradation using MG-132 lead to a rescue of Usp9X and Mcl-1 levels (Fig. 4G). Western blot analyses showed that treatment with G-TPP results in enhanced protein expression of Noxa, a proapoptotic BH3-only Bcl-2 family member, which is known to interact with Mcl-1. We therefore examined next whether Noxa is implicated in G-TPP/ABT263–mediated cell death (Fig. 4H). LN229 glioblastoma cells were transfected with a nontargeting siRNA or two different Noxa-specific siRNAs (Fig. 4H and I). LN229 cells treated with the combination therapy showed a marked reduction of the fraction of sub-G₁ cells in the presence of Noxa knockdown. To assess whether the upregulation of Noxa also results in an increased binding of Noxa to Mcl-1, we performed coimmunoprecipitation experiments (Fig. 4J). LN229 glioblastoma cells were treated with ABT263, G-TPP, or solvent. Subsequently, cells were immunoprecipitated with a monoclonal Mcl-1 antibody and analyzed for the expression of Mcl-1 and Noxa. Although treatment with ABT263 barely affected the binding of Noxa to Mcl-1, treatment with G-TPP lead to a significant increase of the Noxa/Mcl-1 ratio.

LN229 cells treated with increasing concentrations of G-TPP displayed a marked decrease in Mcl-1 and Usp9X levels (Fig. 4K–M; Supplementary Fig. S10). However, in the presence of Noxa knockdown with two different siRNAs this effect was significantly attenuated (Fig. 4K–M; Supplementary Fig. S10).

Upregulation of Noxa is a well-described downstream effect of an unfolded protein response. Previous microarray analyses indicated that low doses of G-TPP cause an unfolded protein response in U251 and LN229 cells, which was accompanied by a transcriptional increase in PMAIP1. In addition, earlier work suggested that interference with mitochondrial matrix chaperones elicits an increase of ER-stress proteins (27). To determine whether a similar upregulation in the unfolded protein response was occurring in our system, we performed Western blot analysis for typical ER-stress markers in LN229 cells treated with increasing concentrations of G-TPP. As shown in Fig. 5A, G-TPP treatment elicits a distinct ER-stress signature, including upregulation of pPERK, CHOP, GRP78, and C/EBPB. On the transcriptional level, these findings were confirmed, showing upregulation of downstream cascade effectors (C/EBPB, XBP1, GRP78, ATF5, and CHOP; Fig. 5B; Supplementary Fig. S8D). To further characterize the link between Noxa upregulation and G-TPP–mediated ER stress, we next examined effects of G-TPP on the integrated stress response. LN229 and U251 cells treated with increasing concentrations of G-TPP showed a dose-dependent increase in p-eIF2α levels, which coincided with a marked increase in ATF4 expression (Fig. 5C and D). To further examine the causal relationship between ATF4 and Noxa levels, LN229 and U251 cells were transfected with a specific siRNA against ATF4. LN229 and U251 cells silenced for ATF4 showed an attenuated upregulation of Noxa after treatment with G-TPP, suggesting that ATF4 plays a central role in G-TPP–mediated upregulation of Noxa (Fig. 5E and F). PERK (protein kinase R-like endoplasmic reticulum kinase) acts as one of the key sensors for the unfolded protein response and is known to activate eIF2α. To extend our understanding on the role of ER stress with respect to G-TPP-mediated ATF4 and Noxa upregulation, we performed knockdown experiments. As we observed before, treatment with G-TPP results in a marked upregulation of ATF4 (Fig. 5G–J). However, in the presence of PERK knockdown, using two different siRNAs ATF4 expression was suppressed despite simultaneous treatment with G-TPP.

We next examined whether a combined inhibition of mitochondrial matrix chaperones and Bcl-2/Bcl-xL yields enhanced therapeutic efficacy in vivo. To this purpose, GBM12 patient-derived xenograft cells were implanted into the right striatum of nude mice and allowed to form tumors prior to randomization into four treatment arms as outlined in Fig. 6A. Animals bearing GBM12 tumors subjected to the combination therapy had significantly prolonged overall survival compared with animals receiving vehicle or single-agent treatments (median survival: control 21d, ABT263 32.5d, G-TPP 31d and ABT263+G-TPP 85d; Fig. 6A). In line with this finding, GBM12 tumor size was markedly reduced in animals treated with the combination therapy as assessed by MRI imaging (Fig. 6B).

We further extended our in vivo studies onto multiple heterotopic glioma models. Mice carrying subcutaneous xenografts of LN229, U251, and U87MG glioma cells were randomized to the respective treatment groups and tumor sizes were measured (Fig. 6C–H). In all models studied, the growth rate of tumors forming in mice that were subjected to treatment with the combination therapy was reduced. The mean tumor size of tumors that formed in animals subjected to the combination therapy was significantly reduced compared with vehicle or single-agent treatments (Fig. 6C–H). Moreover, in all three models, the combined treatment with G-TPP and ABT263 or G-TPP and GX15-070 yielded regression of tumors. Notably, histologic analyses with respect to potential toxic side effects of the combination therapy revealed no detectable noxious effects on solid organs (Supplementary Fig. S11).

For the breast cancer model, MDA-MB-231 cells were implanted into the mammary fat pad of nude mice. Toward the end of the experiment, the mean size of tumors developing in mice that received the combination therapy was significantly reduced compared with vehicle-treated mice (Supplementary Fig. S12). However, despite a clear trend, no statistically significant difference compared with the mean tumor size in the single-agent treatment groups was noted and no regression of tumors was achieved. For the melanoma model, A375 cells were implanted subcutaneously and treatment was initiated as indicated once tumors formed (Fig. 7A–E). Animals subjected to the combination therapy showed a marked reduction of the tumor growth rate culminating in a significantly decreased mean tumor size towards the end of the experiment when compared to single treatments or vehicle (Fig. 7A–C). Histologic analysis revealed a marked decrease in Ki67 staining (Fig. 7D) and a marked increase in TUNEL staining (Fig. 7E) in those tumors forming in animals that received the combination therapy, suggesting that the antitumor effect of the combination therapy is not only due to inhibitory effects on proliferation, but also due to enhanced cell death induction. Treatment with B-Raf^V600E inhibitors represents a mainstay in the treatment of B-Raf^V600E–mutated melanomas. Unfortunately, development of resistance towards B-Raf^V600E inhibitors is a common event. Therefore, we examined whether the combination treatment with ABT263 and G-TPP would prove to be advantageous in a model of B-Raf^V600E inhibitor–resistant melanoma (A375R). In vitro, combined treatment with ABT263 and G-TPP yielded a synergistic anti-proliferative activity in A375R cells (Supplementary Fig. S3B). To test whether this finding also holds up in vivo, A375R cells were implanted subcutaneously and tumors were allowed to form prior to treatment. Animals treated with the B-Raf^V600E inhibitor PLX-4720 displayed only a slight therapeutic response (Fig. 7F–H). However, those animals subjected to treatment with the combination therapy in the presence or absence of PLX-4720 showed a significantly reduced tumor growth rate and mean tumor size toward the end of the experiment (Fig. 7F–H).

High levels of Mcl-1 are known to cause therapeutic resistance towards a certain class of BH3 mimetics (28), which only bind to Bcl-2, Bcl-xL and Bcl-w, but not Mcl-1 (10–14, 29, 30). Mcl-1 is an unstable protein and is regulated by the deubiquitinase Usp9X (31), which is increased in tumors as well, mediates therapeutic resistance and is explored as a potential drug target (22, 31). Researchers have focused to develop strategies to suppress Mcl-1 levels by targeting the different regulatory levels of Mcl-1 expression (12, 18, 22, 24–26, 32–37). Other mechanisms for sensitization to BH3 mimetics include upregulation of the proapoptotic BIM (38, 39).

In this report, we have provided evidence that inhibition of mtHSP90 (2, 6, 18, 40, 41) by G-TPP leads to a reduction of Mcl-1 (42) and its partner Usp9X by eliciting an integrated stress response (ISR) dependent on ATF4 (43–45) with a subsequent increase in proapoptotic Noxa. In turn, Noxa destabilizes both Mcl-1 and Usp9X, sensitizing broadly to the cell death inducing effects of selective and broad BH3 mimetics. Others have described a similar relationship between Noxa and Usp9X after Pemetrexed treatment (46). This is the first report, demonstrating that mitochondrial matrix chaperones control the levels of antiapoptotic Mcl-1. However, based on our results that selective inhibition of Mcl-1 by A-1210477 and siRNA enhances G-TPP–mediated cell death, it is likely that the general tumor-specific mitochondriotoxic properties of G-TPP contributed to the enhancement of BH3-mimetic-mediated cell death as well.

Our findings suggest that this treatment strategy is efficacious in a broad range of in vivo model systems, is well tolerated, and did not reveal any histologically detectable damages to major organ systems, consistent with earlier findings in vivo (5). Therefore, our proposed strategy of targeting Bcl-2 family proteins along with mitochondrial matrix chaperones warrants testing in clinical trials.

No potential conflicts of interest were disclosed.

Conception and design: G. Karpel-Massler, M. Banu, P. Canoll, D.C. Altieri, M.D. Siegelin

Development of methodology: G. Karpel-Massler, P. Canoll, M.D. Siegelin

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): G. Karpel-Massler, C.T. Ishida, R. Perez-Lorenzo, M. Banu, D.C. Altieri, M.D. Siegelin

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): G. Karpel-Massler, C.T. Ishida, E. Bianchetti, C. Shu, P. Canoll, M.D. Siegelin

Writing, review, and/or revision of the manuscript: G. Karpel-Massler, E. Bianchetti, R. Perez-Lorenzo, B. Horst, K.A. Roth, J.N. Bruce, P. Canoll, D.C. Altieri, M.D. Siegelin

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.T. Ishida, C. Shu, B. Horst, M.D. Siegelin

Study supervision: J.N. Bruce, M.D. Siegelin

G. Karpel-Massler received a scholarship from Dr. Mildred Scheel Foundation of the German Cancer Aid. D.C. Altieri was supported by NIH grant NCI P01CA140043, and M.D. Siegelin was supported by the NIH NINDS R01NS095848 and K08NS083732, 2013 AACR-National Brain Tumor Society Career Development Award for Translational Brain Tumor Research (13-20-23-SIEG), BCURED Fighting Brain Cancer Award (16-0992), and American Brain Tumor Association, Translational Grant 2013 (ABTACU13-0098).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.