The development of myeloid and lymphoid neoplasms related to overexpression of FGFR1 kinases as a result of chromosome translocations depends on the promotion of a stem cell phenotype, suppression of terminal differentiation, and resistance to apoptosis. These phenotypes are related to the stem cell leukemia/lymphoma syndrome (SCLL), which arises through the effects of the activated FGFR1 kinase on gene transcription, which includes miRNA dysregulation. In a screen for miRNAs that are directly regulated by FGFR1, and which stimulate cell proliferation and survival, we identified miR-339-5p, which is highly upregulated in cells carrying various different chimeric kinases. Overexpression of miR-339-5p in SCLL cell types enhances cell survival and inhibition of its function leads to reduced cell viability. miR-339-5p overexpression protects cells from the consequences of FGFR1 inactivation, promoting cell-cycle progression and reduced apoptosis. Transient luciferase reporter assays and qRT-PCR detection of endogenous miR-339-5p expression in stably transduced cell lines demonstrated that BCR-FGFR1 can directly regulate miR-339-5p expression. This correlation between miR-339-5p and FGFR1 expression is also seen in primary human B-cell precursor acute lymphoblastic leukemia. In a screen to identify targets of miR-339-5p, we identified and verified the BCL2L11 and BAX genes, which can promote apoptosis. In vivo, SCLL cells forced to overexpress miR-339-5p show a more rapid onset of disease and poorer survival compared with parental cells expressing endogenous levels of miR-339-5p. Analysis of human primary B-cell precursor ALL shows a significant higher expression of miR339-5p compared with the two cohorts of CLL patient samples, suggesting direct roles in disease progression and supporting the evidence generated in mouse models of SCLL.
Significance: Proapoptiotic genes that are direct targets of miR-339-5p significantly influence promotion and aggressive development of leukemia/lymphomas associated with FGFR1 overexpression. Cancer Res; 78(13); 3522–31. ©2018 AACR.
miRNAs provide a mechanism of posttranslational regulation of gene expression either by targeting specific mRNAs for degradation or interfering with their translation into proteins (1). The net effect is the suppression of function for the target genes. miR action is DNA sequence directed, and so individual miRs have the capacity to simultaneously target many genes, providing extensive regulation of the cellular gene expression profile. It is now clear that cancer development is intimately associated with miR regulation of genes involved with proliferation, invasion, and other hallmarks of cancer (2). Analysis of acute myelogenous leukemia (AML), for example, has identified a broad involvement of miRNAs, which can be specific to the particular molecular subtype (3). In our recent studies involving analysis of a stem cell leukemia/lymphoma syndrome (SCLL), expression analysis identified a core group of miRNAs that affected proliferation and survival of the leukemias and lymphomas that have been generated in mouse models of this disease (4).
SCLL develops as a result of the ligand-independent, constitutive activation of FGFR1 kinase as a result of chromosome translocations that lead to chimeric FGFR1 kinases (5). There are a variety of different chromosome rearrangements described in SCLL, but FGFR1 is always involved and in all cases, the partner genes provide the dimerization capability needed for constitutive FGFR1 activation (6). FGFR1 is a transcription factor that regulates specific genes but the fusion kinases also activate cytoplasmic proteins through tyrosine phosphorylation (7). During our screen for miRNAs potentially activated by FGFR1, we identified a series that were downregulated when FGFR1 function was pharmacologically suppressed (4). Multiple members of a closely related family, miR-17/92, were shown to have a significant effect on growth and survival of SCLL cells but, during this analysis, several other individual miRNAs also had profound effects on growth, such as miR-339-5p. Here we establish that miR-339-5p promotes proliferation and survival of SCLL cells through downregulation of the BCL2L11 and BAX proapoptotic genes.
Materials and Methods
Cells and cell culture
BBC2, KG1, and BaF3 cells were cultured in RPMI1640 medium containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin. NIH3T3 and HEK293T cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin. For acid-induced apoptosis, cell lines were grown in standard culture medium, adjusted to pH 5.6 with HCl and then filtered as described previously (8). Cells were then collected for analysis at the indicated time points. Retroviral transduction was performed as described previously (4, 9). In brief, phoenix-ampho packaging cells were transfected with DNA plasmids using Lipofectamine 2000. The viral supernatant was collected after 48 and 72 hours and was used for RetroNectin-mediated infection, following the manufacturer's protocol. Selection with puromycin was performed at a concentration of 1 μg/mL for 4 days, followed by culture in the absence of the selection agent. SCLL cell lines were generated in-house and verified through the expression of the fusion kinases that define them. BaF3 cells are confirmed by virtue of their IL3 dependence, 3T3 cells were obtained from ATCC and used within 5 passages from recovery from frozen. Identity of these cells was confirmed by constant morphology analysis as recommended by ATCC. Mycoplasma testing was not routinely performed.
For overexpression studies, approximately 500-bp fragment encompassing miR-339-5p, including the primary miRNA and flanking sequence, was cloned into pMSCV-PIG (Addgene plasmid #21654). miRNA sponge constructs, which serve as competitive inhibitors of the target miRNA, were generated by multiple insertion of oligoduplexes (Thermo Fisher Scientific) containing 3 bulged miRNA binding sites against miR-339-5p into the pMSCV-PIG-sp vector (10). The promoter region of miR-339-5p was PCR amplified from genomic DNA and inserted into pGL3 luciferase reporter vectors. Oligonucleotides (∼90 bp) containing the original or mutated target sites were cloned into the pMIR-REPORT miRNA expression reporter vector (Thermo Fisher Scientific).
RT-PCR and Western blot analysis
Quantitative RT-PCR and Western blotting assays were carried out as described previously (4).
For the GFP competition assay, cells containing exogenous constructs or empty vector (GFP+) were mixed 50:50 with parental cells (GFP−) and the GFP+/GFP− ratio was determined at different time points using FACSCanto II flow cytometry (BD Biosciences). Cell-cycle profiles were determined with the LSR II flow cytometer (BD Biosciences) following Hoechst 33342 (Thermo Fisher Scientific) labeling. To measure apoptosis, cells were stained with APC Annexin V DNA binding dye (Biolegend) according to the manufacturer's protocol and analyzed using FACSCanto II flow cytometry (BD Biosciences).
Luciferase reporter assay
Promoter assays were performed using HEK293T cells following cotransfection of a promoter reporter plasmid derived from pGL3 with or without pMSCV-BCR-FGFR1 containing plasmids as described previously (4). For miRNA target assays, the luciferase target site-containing plasmids were cotransfected with the miR-339-5p overexpression plasmid into HEK293T cells. The transfected cells were then harvested 48 hours after transfection and analyzed using the Dual-Luciferase Reporter Assay System (Promega). Renilla luciferase was used to normalize for transfection efficiency, and the ratio of firefly/Renilla luciferase activities defined the relative promoter activity. The pMIR-REPORTER system was used to verify miRNA target sites where the predicted target sites were introduced into the multiple cloning site in the 3′ UTR of the luciferase gene (see Results).
In vivo studies
Approximately 1 × 106 cells were injected into the tail veins of 6- to 8-week-old, female, sublethally (600 Rad) irradiated Balb/c mice (9). Disease progression was monitored for the expression of GFP through weekly analysis of peripheral blood derived from the tail vein. At the end of the experiment, mice were sacrificed and organs harvested for evaluation of tumor burden using flow cytometry and morphologic examination. Standard histopathologic analysis of organs was performed as described previously (9, 11). Animal protocols followed guidelines and procedures approved by the Institutional Animal Care and Use Committee of Augusta University.
miR-339-5p is overexpressed and promotes cell growth in SCLL
Suppression of FGFR1 activity using the BGJ398 kinase inhibitor in SCLL cell lines showed significant downregulation of miR-339-5p (4). To analyze the relative expression levels of miR-339-5p in SCLL cells, we confirmed the reported immunophenotypes of three murine cell lines (Supplementary Fig. S1); BBC2, which expresses BCR-FGFR1, was CD19+ IgM−, ZNF112, which expresses ZMYM2-FGFR1, was CD4+CD8+ and BCRF8C, which also expresses BCR-FGFR1, was Sca1+. When miR-339-5p levels were measured in the SCLL cells, compared with flow sorted normal counterparts (Supplementary Fig. S1), there was a highly significant upregulation in all three cell lines (Fig. 1A). In addition, miR-339-5p was also upregulated in FGFR1OP2-FGFR1 expressing human KG1 AML cells (12). When the same four cell lines are treated with BGJ398 (Fig. 1B), which has been shown to suppress chimeric FGFR1 kinase activation in SCLL (13), there is a 50%–80% reduction in miR-339-5p expression levels (depending on the cell line). The same increase in miR-339-5p expression (Fig. 1C) was seen in tumor cells from BCR-FGFR1 and ZMYM2-FGFR1 expressing primary leukemias from murine models (9, 11).
Using the GFP competition assay (see Materials and Methods), cell growth was significantly increased when miR-339-5p was overexpressed in BBC2 cells (Fig. 2A and B). Trypan blue exclusion analysis (Fig. 2C) and luminescence cell viability assays (Fig. 2D) further support this increased growth rate/survival. The same proportional increase in proliferation and viability was seen in the human SCLL KG1 cell line (Fig. 2E–H). To inhibit miR-339-5p, we developed dominant-negative, bulged sponge constructs, which are complementary to miR-339-5p and so, when expressed at high levels, can specifically and efficiently inhibit the miR-339-5p function. When miR-339-5p function was suppressed using these miRNA sponges in BBC2 (Fig. 2I–J), cell proliferation and viability was significantly reduced. Flow cytometric analysis, comparing the control cells with those treated with the sponge constructs, shows significantly increased levels of Annexin V–positive cells, demonstrating increased apoptosis in BBC2 (Fig. 2K). The same reduced proliferation and cell viability was seen in human KG1 cells treated with the miRNA sponges (Fig. 2L and M) and apoptosis was also increased (Fig. 2N). These observations confirm the potential tumor promotion role of miR-339-5p in SCLL.
Overexpression of miR-339-5p mediates resistance to BGJ398-induced cell-cycle inhibition and apoptosis
The BGJ398 FGFR1 inhibitor leads to growth suppression and increased apoptosis in SCLL cells (13). To investigate how miR-339-5p responds to FGFR1 inhibition, BBC2 cells overexpressing either the empty vector (GFP+) or miR-339-5p (GFP+) were mixed 50:50 with the parental cell line (GFP−) and treated with BGJ398 for 72 hours. Cells transduced with the empty vector showed a growth rate approximately equal to the parental BBC2 cells in the presence of BGJ398, whereas cells overexpressing miR-339-5p showed a growth advantage over the parental cells, with increased GFP+ ratios from 50% to 78.7% (Fig. 3A). Flow cytometric analysis of the cell cycle in these cells showed a significant reduction in the proportion of cells in S/G2–M in both the EV-expressing cells and parental BBC2 cells. In contrast, overexpression of miR-339-5p led to a disproportionate increase in the number of cells in S/G2–M compared with the parental cells (Fig. 3A). Thus, overexpression of miR-339-5p can mitigate the cellular effect caused by inhibition of FGFR1 signaling and in a dose-dependent manner (Fig. 3B). In addition to its effect on the cell cycle, miR-339-5p overexpression also mitigated sensitivity to apoptosis induced by BGJ398 (Fig. 3C). Together, these observations suggested a direct involvement of miR-339-5p in FGFR1 fusion kinase–driven disease progression.
miR-339-5p expression is regulated by chimeric FGFR1 kinases
The correlation between expression levels of FGFR1 and miR-339-5p suggested that its expression is regulated by FGFR1. We therefore cloned the miR-339 promoter region (−1626 to −7 bp) into the pGL3 luciferase reporter vector and cotransfected into 293T cells with varying amounts of the BCR-FGFR1 construct. As shown in Fig. 4A, luciferase expression was activated in a dose-dependent manner in the presence of BCR-FGFR1, providing direct evidence that miR-339-5p is activated as a consequence of FGFR1 expression. This suggestion was further supported by the demonstration that NIH3T3 and BaF3 cell lines, stably transduced with BCR-FGFR1, also showed increased expression of miR-339-5p (Fig. 4B). The relationship between FGFR1 and miR-339-5p expression was further supported by an analysis of miRNA and mRNA expression profiles in a cohort of patients with B-cell precursor acute lymphoblastic leukemia (ALL), which revealed a significant, positive correlation between the expression of FGFR1 and miR-339-5p (Fig. 4C). There was no correlation in a similar analysis of two cohorts of patients with relatively benign CLL, possibly due to relative low expression levels of miR-339-5p in these leukemias (Supplementary Fig. S2A–C). A similar comparison between B-cell precursor ALL and datasets for either normal donor B cell or CLL samples, showed a significantly higher expression of miR-339-5p (Fig. 4D; Supplementary Fig. S2D) compared with the CLL patients when normalized either with housekeeping miR-16 or Let-7a. In contrast, no differential expression for control housekeeping miRNA Let-7a and miR-16-5p was seen in these cohorts (Supplementary Fig. S2E and F). Together, these data support the importance of miR-339-5p in chimeric FGRF1-induced SCLL.
miR-339-5p regulates activity of BCL2L11 and BAX
The demonstration that miR-339-5p promotes cell viability in SCLL cells, suggested that it targets transcripts related to this phenotype. Bioinformatics analysis, using sequence-based prediction algorithms, including TargetScan and miRWalker2.0 (14, 15), were used to identify potential miR-339 mRNA targets (Supplementary Fig. S3; Supplementary Table ST1). Because miR-339-5p appears to affect apoptosis (Fig. 3), we filtered genes that affected this phenotype and identified BCL2L11 and BAX as candidate targets (Fig. 5A and B; Supplementary Fig. S3). Target sites in these genes were identified in the 3′ UTR of BCL2L11 and in the coding region of BAX. To determine whether miR-339-5p directly regulates BCL2L11 and BAX expression, we performed luciferase reporter assays where the target sites, with approximately 90-bp flanking sequences, were cloned into the pMIR-REPORT luciferase reporter vector. When 293FT cells were cotransfected with miR-339-5p and luciferase constructs, there was a 50% reduction in luciferase activity of both BCL2L11 and BAX in the presence of miR-339-5p (Fig. 5C and D). Mutation of the target sites (Fig. 5A and B) in each case abrogated the suppressive effect of miR-339-5p. Western blot analysis showed that miR-339-5p overexpression reduced BCL2L11 and BAX proteins levels, and miRNA sponges targeting miR-339-5p led to an increase in BCL2L11 and BAX protein levels in both BBC2 and KG1 cells, further confirming that BCL2L11 and BAX were direct targets of miR-339-5p (Fig. 5E). Analysis of primary SCLL spleen cells (Fig. 5F) from both the BCR-FGFR1 murine model and the BCR-FGFR1 human cell model demonstrated downregulation of BCL2L11 and BAX compared with normal splenic cells (Fig. 5G; refs. 11, 16). These data indicate that miR-339-5p negatively regulates BCL2L11 and BAX in SCLL.
To further validate the specificity of miR-339-5p for the predicted target site in the 3′ UTR of BCL2L11, we used the pMIR-REPORT Luciferase assay and introduced these BCL2L11 target sites into the 3′ UTR (Supplementary Fig. S4A). In this assay, miRNA targeting of these sites leads to destabilization of the luciferase mRNA, resulting in reduced activity. When the luciferase reporter was cotransfected into 293T cells with the miR339-5p expression vector, luciferase activity was highly significantly reduced (Supplementary Fig. S4B). Next, we prepared oligonucleotides with 3–9 repeats of the miR339-5p target sequence in the pMCSV-PIG-Sp vector (Supplementary Fig. S4A) and cotransfected these into 293T cells together with the miR339-5p expression vector and the luciferase gene harboring the BCL2L11 target sites. It was anticipated that the introduced miR339-5p target sites would compete for miR339-5p binding and so suppress the effect on luciferase mRNA degradation. As shown in Supplementary Fig. S4B, luciferase activity in the presence of the multiple binding site constructs increased proportionally to the number of tandem binding sites, further supporting the specificity of miR339-5p for the BCL2L11 target sites. To further support the specific effects of the sponge constructs in suppressing miR339-5p, we again used the luciferase-BCL2L11 reporter assay where the miR339-5p expression construct was cotransfected with different ratios of the sponge constructs (Supplementary Fig. S4C) where the higher the concentration of sponges correlated with increased luciferase activity. These data provide further evidence that the sponge constructs can efficiently target and suppress miR-339-5p activity.
To broaden the context of the effects of miR-339-5p, we analyzed the relative mRNA expression levels of miR-339-5p, BAX and BCL2L11 in a dataset (GSE79547) available in GEO for B-cell precursor ALL. No significant correlation (Fig. 5H) was observed in the expression levels of BAX and miR-339-5p, which might have been expected since miR-339-5p interferes with protein translation rather than mRNA stability. In contrast, there is a highly significant correlation between miR-339-5p and BCL2L11 expression in this analysis, where higher levels of miR-339-5p led to reduced levels of BCL2L11, which is expected as miR-339-5p targets sequences in the 3′UTR affecting mRNA stability as described above. This observation supports the idea that miR-339-5p can promote leukemogenesis and SCLL progression through targeting BAX and BCL2L11.
miR-339-5p protects SCLL cells from induced apoptosis
Reduced apoptosis as a result of miR-339-5p overexpression in BGJ398-treated cells, as well as miRNA target analysis identifying BCL2L11 and BAX, suggested that miR-339-5p could promote SCLL through protection against apoptosis. To investigate this hypothesis, we induced apoptosis using reduced acidic conditions. When miR-339-5p–overexpressing cells were placed in pH5.6 culture conditions, there was a significant increase in cell viability, compared with cells transfected with the empty vector alone (Fig. 6A and B). Cell viability was again reduced when miR-339-5p sponges were introduced into the cells. When the BCL2L11 overexpression construct that lacked miR-339-5p target sites in the 3′UTR was coexpressed in cells overexpressing miR-339-5p, cell survival was also reduced in the low pH environment (Fig. 6A and B). Analysis of the Annexin V levels in these different cell lines demonstrated that reduction in cell viability was consistent with increased levels of apoptosis (Fig. 6C; Supplementary Fig. S5A). These observations support the conclusion that miR-339-5p can directly target proapoptotic BCL2L11 and affect apoptosis of SCLL cells. This conclusion is further supported by the fact that the same effects on apoptosis were seen when cells were treated with BGJ398 (Fig. 6C; Supplementary Fig. S5B).
At the protein level, the reduced apoptosis phenotype was accompanied by an impaired activation of BCL2L11 and BAX in the miR-339-5p–overexpressing cells, compared with the empty vector control (Fig. 6D). The resistance of miR-339-5p–expressing cells to acid induced apoptosis was also confirmed by the delay in the activation and cleavage of PARP (Fig. 6D), a hallmark of apoptosis (17). These data demonstrate miR-339-5p can protect cells from apoptosis by targeting BCL2L11 and BAX.
miR-339-5p enhances SCLL development in vivo
To evaluate the oncogenic effect of miR-339-5p in FGFR1 fusion kinase–driven leukemogenesis in vivo, 1 × 106 BBC2 cells, either stably overexpressing miR-339-5p or the vector control, were injected into the tail veins of 6- to 8-week old (n = 5), sublethally irradiated, syngeneic BALB/c mice as described previously (9). Consistent with the in vitro cell proliferation effects, overexpression of miR-339-5p increased the proportion of GFP+ BBC2 cells in the peripheral blood from 20.29% to 40.33% (P = 1.75e−03) one week after transplantation (Fig. 7A). Aggressive disease subsequently developed in miR-339-5p–overexpressing cells within 10–25 days (Supplementary Fig. S6A and B) and Kaplan–Meier comparison between mice injected with the miR-339-5p–overexpressing BBC2 cells and parental BBC2 cells, shows a highly significant (P = 0.004) reduction in survival (Fig. 7B). In addition, the mice transplanted with the BBC2 cells with forced expression of miR-339-5p displayed reduced body weight due to disease burden (Fig. 7C), as well as significantly enlarged spleens (Fig. 7D) and livers (Fig. 7E), compared with empty vector control cells. Consistently, the white blood cell count in the miR-339-5p group on termination of the experiment was significantly higher than for the empty vector control group (Fig. 7F). Flow cytometry analysis of cells in the bone marrow and spleens from mice engrafted with BBC2 that express either the empty vector or miR-339-5p showed that the vast majority were GFP+, and the more enlarged spleens from the miR-339-5p–overexpressing group had an even higher ratio of GFP+ cells (Fig. 7G). Histopathology analysis confirmed these findings, with increased cellularity in the peripheral blood of mice overexpressing miR-339-5p, which was associated with hypercellularity in the bone marrow and increased infiltration into the spleen and liver (Supplementary Fig. S6C).
FGFR1 activation has been suggested to lead to extensive gene regulation, either directly though promoter binding or indirectly through the activation of other cofactors such as CBP, that activate a diverse series of transcription factors (7). Here we show that FGFR1 can also regulate specific miRNA expression profiles, which adds to the complexity of the transformation process through posttranslational modification of gene function. Some miRNAs detected in our screen appear to have more profound effects on growth and survival, presumably as a result of the genes they regulate. Maintenance of an undifferentiated state is observed in SCLL development (18), as well as suppression of apoptosis. In this respect, the ability of miR-339-5p to suppress the function of genes that promote apoptosis directly affects survival of the tumor cells. miR-339-5p has previously been shown to affect expression of genes such as PRL1 (19), and many others through prediction algorithms that have not been functionally confirmed.
Here we show for the first time that miR-339 can affect production of BAX and BCL2L11. BAX interacts with the BCL2 apoptosis suppressor to promote metastasis through destabilization of the mitochondrial membrane promoting release of cytochrome c. BCL2L11, also known as BIM or BimEL, which is prominently expressed in cells of the hematopoietic linage (20), is an essential proapoptotic protein involved in the initiation of apoptotic pathways by binding to antiapoptotic proteins of the BCL2 family (21). Growth factors such as IL3 promote survival of hematopoietic progenitors through downregulation of BCL2L11, and this IL3-dependent downregulation is mediated through activation of Raf/MAPK and PI3K/mTOR pathways (22). BCL2L11 is also important for determining the lifespan of myeloid and lymphoid cells, as these cells show increased numbers in mice lacking BCL2L11 (23). BCL2L11 can also be targeted by several other oncomiRs, such as miR-17–92, miR-106–25, miR-181, miR-148, and miR-221/222, which promote tumor progression (21). Here, we identified a new oncomiR, miR-339-5p, which promotes SCLL through double targeting proapoptotic genes BCL2L11 and BAX. Interestingly, BCL2L11 is also regulated by miR-17-92, which we have shown is also upregulated in FGFR1-driven SCLL (4), suggesting FGFR1 fusion kinases simultaneously regulate multiple miRNAs to inhibit key apoptotic genes to promote leukemogenesis.
Although data from SCLL suggests that miR-339-5p strongly promotes cell survival, other studies using MCF7 breast cancer cells (24), for example, showed miR-339-5p could influence levels of p53. This effect was mediated through its targeting of MDM2, thus having an antiproliferation effect while apparently not affecting apoptosis. Correlative studies in other breast cancer models also suggested a role for miR-339-5p in the suppression of invasion and metastasis (25). Similar conclusions were reached in colon cancer (26); in this case though, its regulation of PRL1. Different miRNAs can be generated from the same pre-miRNA, but often have different effects and different targets. Thus, the alternatively derived miR-339-3p has also been reported as a suppressor of invasion in melanoma cells with no effect on cell proliferation (27). In this latter case, MCL1 appears to be a target, which also has a role in promoting apoptosis. The clear ability of miR-339-5p to promote survival in FGFR1-expressing myeloid and lymphoid malignancies, in contrast to the opposite effect seen in cells from solid tumors, suggests a cell context–specific action.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: T. Hu, J.K. Cowell
Development of methodology: T. Hu, Y. Chong, S. Lu, C.-S. Chang
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Hu, Y. Chong, S. Lu, R. Wang, H. Qin, E. Kitamura
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Hu, Y. Chong, J. Silva, C.-S. Chang, L.A. Hawthorn, J.K. Cowell
Writing, review, and/or revision of the manuscript: T. Hu, J. Silva, J.K. Cowell
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Hu, Y. Chong, S. Lu, E. Kitamura, J.K. Cowell
Study supervision: T. Hu, J.K. Cowell
This work was supported by grant CA076167 from the NIH (to J. Cowell).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.