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[Cancer Research 60, 6178-6183, November 1, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

Tumor Induction by the c-Myc Target Genes rcl and Lactate Dehydrogenase A1

Brian C. Lewis2, Julia E. Prescott, Susan E. Campbell, Hyunsuk Shim, Robert Z. Orlowski3 and Chi V. Dang4

Program in Human Genetics and Molecular Biology [B. C. L., J. E. P., C. V. D.], Departments of Medicine [B. C. L., J. E. P., S. E. C., H. S., R. Z. O., C. V. D.] and Molecular Biology and Genetics [C. V. D.], and Johns Hopkins Oncology Center [R. Z. O., C. V. D.], The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The characterization of c-Myc target genes, such as rcl and lactate dehydrogenase A (LDH-A), is critical for understanding the mechanisms of c-Myc-induced cell transformation and tumorigenesis. We have previously demonstrated that Rcl induces anchorage-independent growth in Rat1a fibroblasts and that LDH-A is required for cell transformation by c-Myc. In this study, we report that Rcl and LDH-A act synergistically to induce anchorage-independent growth. Cells expressing both Rcl and LDH-A form tumors after s.c. injection into nude mice, although neither Rcl or LDH-A overexpression alone induces tumorigenesis. The inability of Rcl and LDH-A to fully recapitulate c-Myc activity, however, indicates that other c-Myc target genes participate in tumorigenesis. In addition, cells that coexpress Rcl and vascular endothelial growth factor are more comparable with c-Myc overexpressing cells in their ability to form tumors in nude mice. These findings confirm Rcl and LDH-A as critical components of the cell transformation program induced by c-Myc and suggest that Rcl is tumorigenic in cells that are provided with a permissive metabolic milieu.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The c-myc oncogene was first identified as the cellular homologue of the transforming viral oncogene v-myc (1) . Deregulated expression of the c-myc oncogene by gene amplification and other mechanisms is a common feature of human malignancies (2, 3, 4, 5) . Translocation of the c-myc locus on chromosome 8 that fuses it to regulatory elements of the immunoglobulin heavy chain is the hallmark of Burkitt lymphoma (6 , 7) . Recent data demonstrating that the c-myc gene may be a target of the adenomatous polyposis coli pathway, which is frequently disrupted in colon carcinoma, further illustrate the significance of c-myc alterations in human malignancy (8) .

The protein product of the c-myc gene, c-Myc, is a basic helix-loop-helix-leucine-zipper (bHLHzip) transcription factor. c-Myc binds to its partner bHLHzip protein Max, and this heterodimer binds to specific DNA sequences and regulates transcription (9 , 10) . c-Myc transcriptional activity has been shown to be involved in the regulation of cell proliferation, cell transformation, and apoptosis (11, 12, 13) . Genes transcriptionally regulated by c-Myc are, therefore, hypothesized to encode proteins involved in these processes. The identification and characterization of c-Myc transcriptional targets will, therefore, allow greater understanding of the mechanisms by which c-Myc affects these cellular processes and increase the understanding of the mechanisms by which c-Myc contributes to human malignancies.

We have previously described the use of representational difference analysis to identify a collection of putative c-Myc-responsive genes in Rat1a fibroblasts. In that report we described the characterization of a novel c-Myc target gene, termed rcl (14) . The expression of rcl is very low in untransformed cell lines. The rcl mRNA levels are, however, significantly elevated in lymphoma and breast carcinoma cell lines (14) .5 Furthermore, Serial Analysis of Gene Expression studies reveal that rcl expression is elevated in human glioblastoma multiforme as compared with the very low expression in normal human brain (this information is available through the UniGene database).6 Rcl is a Mr 23,000 nuclear protein of unknown function, the expression of which is growth related. Ectopic Rcl expression induces anchorage-independent growth in Rat1a fibroblasts (14) . We have also identified the LDH-A7 gene as a direct c-Myc transcriptional target (15) . LDH-A is responsible for the conversion of pyruvate to lactate during glycolysis. LDH-A expression is necessary for c-Myc-mediated anchorage-independent growth, and elevated LDH-A expression underlies a novel c-Myc-mediated apoptotic pathway induced by glucose deprivation (15 , 16) . Elevated LDH-A expression may underlie the ability of tumor cells to undergo aerobic glycolysis, the so-called Warburg effect (15) .

On the basis of our previous studies of Rcl, and the known role of LDH-A in glycolysis, we hypothesized that ectopic Rcl expression stimulates progression through the cell cycle. We also surmised that deregulated LDH-A expression provides a metabolic environment compatible with growth in a three-dimensional multicellular sphere and that Rcl and LDH-A might cooperate in the induction of the c-Myc phenotypes of cell transformation, and apoptosis.

We report that Rcl and LDH-A are able to cooperate in the induction of anchorage-independent growth and in tumor formation in nude mice. In support of our hypothesis that Rcl has in vivo transforming activity, we also demonstrate that VEGF can substitute for LDH-A and cooperate with Rcl in the induction of tumors in nude mice. In contrast to the cell transformation and tumorigenic phenotypes, Rcl and LDH-A do not cooperate in the induction of apoptosis. These data represent the first demonstration of cooperation between c-Myc targets in vivo and confirm that Rcl and LDH-A are components of key pathways stimulated by c-Myc in the induction of tumorigenesis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids.
The plasmids pSG5-rcl and pSG5-LDH have been described previously (14 , 15) . The plasmid pSG5-VEGF189 was generated by the excision with BamHI and HindIII of the cDNA encoding the 189 amino acid isoform of VEGF from pCEP4 (a gift from Webster Cavenee, University of California–San Diego, La Jolla, CA) and insertion into a pSG5 vector modified to contain the pBluescript polylinker.

Cell Lines.
Rat1a, Rat1a-rcl, Rat1a-LDH, and Rat1a-myc cell lines have been described previously (14 , 15 , 17 , 18) . Unless otherwise indicated, these cell lines were cultured in DMEM with 10% FBS, penicillin (100 units/ml), and streptomycin (100 mg/ml). Stable cell lines were generated by Lipofectin-mediated transfection for 6 h in OptiMEM (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s protocol. RLR and RLP cell lines were generated by Lipofectin transfection of Rat1a-LDH cells (15) with pSG5-rcl and pSVneo or pSG5 and pSVneo, respectively, at a 20:1 ratio. Pools of resistant cells were selected in DMEM containing 400 µg/ml G418 sulfate. Rat1a-VEGF189 and Rcl-VEGF189 lines were generated by cotransfection of pSG5-VEGF189 and pSVneo into Rat1a or Rat1a-rcl cells at a 20:1 ratio. Pools of resistant cells were selected in DMEM + 400 µg/ml G418 sulfate.

Southern blot analysis of Rat1a, Rat1aVEGF189, Rcl, Rcl-VEGF189, and RLR cell lines shows a dominant rcl hybridizing band that persists in all Rcl-derived Rat1a cell lines (data not shown). It is not detected in either the Rat1a or Rat1aVEGF189 cell lines. This analysis indicates that a dominant Rcl-expressing Rat1a clone exists and that no clonal selection occurs with subsequent coexpression of either LDH or VEGF.

Northern Blot Analysis.
Total RNA was isolated from the indicated cell lines using Trizol reagent (Life Technologies, Inc.) according to the manufacturer’s protocol. Total RNA (10 µg) was subjected to agarose gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was then sequentially probed with a full-length VEGF cDNA probe and a murine ribosomal protein rpL32 cDNA (a gift from R. Muschel, University of Pennsylvania, Philadelphia, PA) as a control. A phosphorimager (Molecular Dynamics, Sunnyvale, CA) was used for signal quantitation.

Immunoblotting.
Cultured cells were washed once with PBS, scraped in western scraping buffer [40 mM Tris-HCl (pH 7.4), 1 mM EDTA (pH 8), 150 mM NaCl], then lysed in 10% SDS. Relative protein amounts were determined using the BCA kit (Pierce Chemical Co., Rockford, IL). Equal amounts of protein were loaded per lane and subjected to SDS PAGE. Immunoblotting was then performed as described previously using enhanced chemiluminescence (Ref. 14 ; Amersham Corp., Arlington Heights, IL). The rabbit polyclonal anti-Rcl antibody was used at 1:1250 dilution (14) . The ß-actin antibody (Sigma Chemical Co., St. Louis, MO) was used at a 1:1000 dilution.

LDH Enzyme Assay.
Cell lysates in 15 mM KCl, 10 mM Tris-HCl (pH 7.4), 1.5 mM MgCl2, and 6 mM ß-mercaptoethanol were centrifuged at 16,000 x g for 5 min, and LDH activity was determined using an LDH enzymatic kit (Sigma Chemical Co.; Ref. 15 ).

Cell Cycle Analysis.
Cell lines were grown for 48 h either on tissue culture dishes or on plates coated with 0.7% agarose, as described previously (19) . After incubation for 30 min with the nucleotide analogue BrdUrd (10 mM), the cells were washed in PBS and fixed in 70% ethanol at -20°C. Cell cycle analysis was then performed as described previously (14) .

Soft Agar Assays.
Cells (1.2 x 105 ) of either Rat1a, Rat1a-myc, RLR, or RLP cell lines in 2-fold concentrated DMEM and 20% FBS were mixed with an equal volume of 0.8% agarose and poured onto a bed of 0.7% agarose, as described previously (20) . Cells were fed every 3 days with 2 ml of DMEM containing 10% FBS. After 12–14 days, photomicrographs were taken and colonies greater than 100 µm in diameter were counted. Experiments were repeated three times.

Apoptosis Assays.
Cells (5 x 105 ) from the above cell lines were plated in DMEM with 10% FBS and allowed to grow for ~24 h. Cells were then washed twice with PBS; refed with media containing either 10% FBS, 0.1% FBS, or glucose-free DMEM media containing 10% FBS; and incubated for 18–20 h, as described previously (16 , 18) . DNA fragmentation during apoptosis was quantified using two-dimensional flow cytometry (21) . Cells were fixed in 1% formaldehyde, 70% methanol, and then washed and incubated at 37°C with the deoxynucleotide analogue biotin-16-dUTP and terminal deoxynucleotidyl transferase (Boehringer Mannheim, Indianapolis, IN). Cells were then treated with FITC-conjugated avidin (Boehringer Mannheim), stained with propidium iodide, and analyzed by flow cytometry as described above for cell cycle analysis.

Nude Mouse Assays.
Cells (5 x 106 ) in 200 µl of sterile PBS from the indicated cell lines were injected s.c. into the right flank of male homozygous nude mice, 4–6 weeks of age (Taconic, Germantown, NY). Tumors were allowed to establish for 6 weeks, or until the estimated tumor mass exceeded 1500 mg, and the mice were sacrificed and tumors removed. Tumors were weighed and either frozen or fixed in formalin. Fixed tumors were sectioned and stained with H&E. Experiments were approved by The Johns Hopkins School of Medicine Animal Care and Use Committee.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We have previously reported that ectopic expression of Rcl in Rat1a fibroblasts leads to the induction of anchorage-independent growth, albeit weakly in comparison with c-Myc (14) . Ectopic expression of LDH-A is insufficient to induce anchorage-independent growth in these cells. However, antisense reduction of LDH-A levels ablates the ability of c-Myc to induce this transformation phenotype (15) . From these data, and the known role of LDH-A in glycolysis, we hypothesized that Rcl expression stimulates growth and that LDH-A expression permits cell proliferation under hypoxic conditions found in a three-dimensional multicellular sphere. We, therefore, investigated whether the c-Myc targets rcl and LDH-A could cooperate in the induction of cell transformation.

A stable cell line, RLR, was generated in which both rcl and LDH-A are expressed in pSG5 plasmid vectors. Rcl expression in the RLR cell line is comparable with that found in Rat1a-rcl cells by immunoblot, when normalized to ß-actin (Fig. 1)Citation . LDH-A enzymatic assays demonstrated that the level of LDH-A enzymatic activity in the RLR line is comparable with that found in Rat1a-LDH lines (Fig. 1B)Citation . A control LDH-A cell line in which the pSG5 vector was transfected (RLP) was also generated. We then compared the ability of the RLR and RLP cell lines to undergo anchorage-independent growth relative to Rat1a-myc cells. The RLR line exhibited anchorage-independent growth, forming colonies at ~48% of the efficiency of Rat1a-myc cells (Fig. 2)Citation . This transformation efficiency is >4-fold that seen with ectopic expression of Rcl alone (14) . Ectopic LDH-A expression alone does not induce anchorage-independent growth, as demonstrated by the RLP cell line. Hence, Rcl and LDH-A do cooperate in the induction of anchorage-independent growth.



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  		Fig. 1. Expression of Rcl protein and LDH-A enzyme activity in Rat1a cell lines. A, immunoblot demonstrating Rcl protein levels in Rcl- and LDH-A-expressing cell lines. The cell line RLP constitutively expresses LDH-A, and the cell line RLR expresses both LDH-A and Rcl. Actin was used as a loading control. Relative Rcl protein levels normalized to actin are: Rat1a, 1.0; RLP, 0.9; Rcl, 17.9; RLR, 12.9; Rat1a-myc, 1.6. B, LDH enzyme activity in the same cell lines.

 


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  		Fig. 2. RLR cells demonstrate anchorage-independent growth. Soft agar assay demonstrating enhanced clonogenicity and colony size of the RLR cell line compared with the Rat1a-rcl and RLP cell lines. Cells were plated in soft agarose, and photomicrographs (magnification, x10) of representative colonies were taken after 2 weeks. Average colony numbers (with SEs shown) from triplicate experiments were: Rat1a, 0; RLP, 99 ± 84; Rcl, 425 ± 120; RLR, 1977 ± 200; and Rat1a-myc, 4155 ± 1325.

 
Ectopic Rcl expression in Rat1a cells, while inducing anchorage-independent growth, does not lead to increased apoptosis when cells are deprived of serum or glucose (14) . Ectopic LDH-A expression has no effect on apoptosis levels when cells are deprived of serum (16) . However, LDH-A expression seems to underlie the induction of apoptosis by c-Myc when cells are deprived of glucose (16) . We, therefore, sought to determine whether Rcl and LDH-A could synergize in the induction of apoptosis. RLR, RLP, Rat1a, and Rat1a-myc cells were either grown in medium containing serum and glucose or exposed for 20 h to medium deficient in either serum (0.1% serum) or glucose (no glucose). When deprived of serum, the Rat1a, RLR, and RLP lines all arrested and displayed low levels of apoptosis, whereas the Rat1a-myc cells displayed significant apoptosis (Fig. 3)Citation . When the cells were deprived of glucose, the LDH-A-expressing lines RLR and RLP displayed levels of apoptosis comparable with that seen in the Rat1a-myc cells, as described previously (16) . The elevated expression of Rcl in the RLR cell line did not lead to either enhanced or reduced levels of apoptosis, suggesting that Rcl expression has no effect on apoptosis in response to these stimuli. These results confirm our earlier hypothesis that c-Myc-induced transformation and apoptosis occur through independent mechanisms and that Rcl is involved in the former but not the latter.



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  		Fig. 3. Apoptosis induced in RLR cells. Cells from the indicated cell lines were cultured in regular medium (top), in the absence of serum (middle), or in the absence of glucose (bottom). Cells were harvested, DNA strand breaks were labeled with biotin-dUTP by terminal deoxynucleotidyl transferase, and total DNA was stained with propidium iodide. DNA content determined by propidium iodide staining is shown on the abscissa, and DNA strand breaks are shown on the ordinate. The numbers underneath each panel represent the percentage of apoptotic cells. Data are shown for a representative experiment. Similar data were obtained in multiple experiments.

 
Having observed that Rcl and LDH-A can cooperate in a cell culture-based transformation assay, we then sought to determine whether they could cooperate in vivo. The ability of Rat1a cells expressing Rcl, LDH-A, or the two together to induce tumors in nude mice was evaluated. Rat1a cells expressing either Rcl or LDH-A alone were poorly tumorigenic in the nude mice, and their rate of tumor induction was similar to that of the control Rat1a cells (Table 1)Citation . By contrast, RLR cells induced tumors in 60% of the animals injected with an average mass of 286 ± 72 mg. Rat1a-myc cells induced large tumors in all of the mice injected with an average mass of 983 ± 196 mg. These data support a correlation between the ability of Rat1a-based cell lines to grow in soft agar and their ability to form tumors in nude mice and further suggest that Rcl and LDH-A are important for c-Myc-induced tumorigenesis in vivo.


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  		Table 1 Tumor induction by Rcl- and LDH-A-expressing cell lines

 
If ectopic Rcl expression allows aberrant cell cycle progression and deregulated LDH-A expression potentiates that effect by providing a metabolic environment that allows growth under hypoxic conditions, then other molecules that provide cells with a permissive metabolic environment, or that induce angiogenesis, such as VEGF, might also cooperate with Rcl in the induction of tumors in vivo. We, therefore, created cell lines in both Rat1a and Rat1a-rcl cells that express either the 121, 165, or 189 amino acid isoforms of VEGF (22) . These VEGF isoforms are all biologically active, but the 121 and 165 amino acid isoforms are freely diffusable molecules, whereas the 189 amino acid isoform is associated with the cell membrane and extracellular matrix (22) . By immunoblot, all Rcl-VEGF cell lines express levels of Rcl similar to the Rat1a-rcl cell line (Fig. 4A)Citation . VEGF expression was determined by Northern blot analysis, which revealed variable mRNA levels for the three VEGF isoforms, with the 189 amino acid isoform most highly expressed (Fig. 4B)Citation . The reasons for the difference in expression of the VEGF isoforms are unclear. The level of expression was similar in both the Rat1a and Rat1a-rcl background (Fig. 4B)Citation .



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  		Fig. 4. Rcl and VEGF expression in Rat1a cell lines. A, immunoblot demonstrating Rcl protein levels in VEGF-expressing cell lines. Actin was used as a loading control. B, Northern blot demonstrating VEGF mRNA levels in the indicated cell lines. rpl32 was used as a loading control.

 
Because the VEGF 189 isoform gave the highest expression, we tested the ability of Rat1a (Rat1a-VEGF189) and Rat1a-rcl (Rcl-VEGF189) cell lines expressing this isoform to generate tumors in nude mice. Tumors were detected in 14 of 15 mice that received injections of Rcl-VEGF189 cells (Table 2)Citation . These tumors were significantly larger in size (average mass, 1072 ± 313 mg) than those produced by the RLR cells but were, on average, smaller than those induced by Rat1a-myc cells (average mass, 1883 ± 301 mg). Only 2 of 15 mice that received injections of Rat1a-VEGF189 cells developed a detectable small tumor (Table 2)Citation . Furthermore, Rat1a-VEGF189 cells did not form colonies in soft agar at a frequency above that of a Rat1a-pSG5 control, and Rcl-VEGF189 cells did not display an enhanced cloning efficiency to that previously described for Rat1a-rcl cells (data not shown). These observations suggest a collaboration of Rcl with VEGF in tumorigenesis in vivo.


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  		Table 2 Tumor induction by Rcl- and VEGF-expressing cell lines

 
Because of the concern that ectopic Rcl expression in the Rat1a cells exceeds the level of Rcl in Rat1a-Myc cells, we determined whether overexpression of another c-Myc target gene could also collaborate with VEGF189 in tumorigenesis. We have identified another c-Myc target gene that encodes a novel nuclear protein, termed JP1.8 Although JP1 overexpression induces anchorage-independent growth of Rat1a cells in vitro, overexpression of JP1 along with VEGF189 in Rat1a cells did not cause tumorigenesis in nude mice (data not shown). These data suggest that VEGF can specifically cooperate with Rcl, but not with JP1, in the induction of tumors in nude mice.

Histological examination of the tumors produced by the Rat1a-myc, RLR, and Rcl-VEGF189 cell lines demonstrated some intriguing differences. The tumors from Rat1a-myc and RLR mice were solid throughout with only modest evidence of necrosis, whereas those from the Rcl-VEGF189 mice displayed highly necrotic centers, accompanied by an infiltrate of eosinophils and neutrophils, with a margin of intact tumor surrounding the necrotic center (Fig. 5)Citation . This was observed in all tumors generated by Rcl-VEGF189 cells. This finding suggests that the elevated expression of LDH-A in the Rat1a-myc and RLR tumors may be protective against cell death induced by the hypoxic conditions present in the center of these tumors.



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  		Fig. 5. Histology of Rat1a-myc, RLR, and Rcl-VEGF189 tumors. H&E-stained sections of tumors from the indicated cell lines at x2 magnification (left) and x40 magnification (right). Areas of necrosis are lightly stained. High magnification images show the presence of inflammatory cells and necrosis in Rcl-VEGF189 (Rcl 189) tumor.

 
Both the Rat1a-myc and Rcl-VEGF189 tumors were highly vascular. In the case of the Rcl-VEGF189 tumors, the majority of the increased vasculature was present along the periphery as demonstrated by the increased staining for von Willebrand factor, a vascular endothelial cell marker (data not shown). This finding suggests that angiogenesis is induced in the Rat1a-myc cells. This process may involve other angiogenic factors such as basic fibroblast growth factor, because VEGF levels in Rat1a-myc cells and tumors are only moderately increased over that seen in Rat1a and RLR cells (data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The characterization of c-Myc transcriptional targets is critical for a greater understanding of the molecular mechanisms of cell transformation. We have previously reported the identification of a collection of c-Myc-responsive genes in Rat1a fibroblasts (14) . Among the putative c-Myc-responsive genes identified in that study, rcl and LDH-A stood out because of their high differential expression between nonadherent Rat1a-myc and Rat1a cells, and because they behave as direct transcriptional targets of c-Myc (14 , 15) . In addition, both of these proteins were shown to be important for c-Myc-induced anchorage-independent growth in Rat1a cells Myc (14 , 15) . Ectopic expression of either Rcl or LDH-A in Rat1a cells recreates different c-Myc phenotypes, suggesting that these proteins act in different pathways (14, 15, 16) . These data prompted us to determine whether these two proteins could synergize in the induction of the c-Myc phenotypes of anchorage-independent growth and apoptosis.

A pooled cell line that constitutively expresses both Rcl and LDH-A (RLR) was tested for its ability to undergo anchorage-independent growth in a soft agar assay. The RLR cells demonstrated a significant transforming potential, forming colonies greater than 100 µm in diameter, at ~40% the rate of Rat1a-myc cells. This transforming potential was greater than the sum of the transforming ability of Rcl and LDH-A individually in Rat1a cells, suggesting that Rcl and LDH-A do, in fact, synergize in the induction of anchorage-independent growth (14 , 15) . It should be noted, however, that the RLR pooled cell lines are still less transformed than the Rat1a-myc cells. Hence, the synergy between Rcl and LDH-A requires additional Myc target genes to recapitulate c-Myc-transforming activity. This is the first observation of synergy between c-Myc target genes and confirms the importance of Rcl and LDH-A in the transformation of Rat1a cells by c-Myc. This demonstration is significant because although some putative c-Myc targets have been implicated in cell transformation and tumorigenesis, several others, such as {alpha}-prothymosin and ECA39, do not induce transformation phenotypes when overexpressed (23, 24, 25, 26, 27, 28, 29, 30) . Our data suggest that these proteins may require coexpression of other c-Myc targets, for the induction of transformation phenotypes. The ability of putative c-Myc target genes to synergize in the induction of cell transformation and apoptosis may, therefore, be used to identify those targets that play critical roles in biological processes regulated by c-Myc.

Subcutaneous injection of RLR cells, which coexpress Rcl and LDH-A, into nude mice induced tumors >100 mg in mass in 60% of the mice. In contrast, cells expressing either Rcl or LDH-A alone did not produce tumors at a frequency higher than untransfected Rat1a cells. Tumors occurred in 100% of mice that received injections of Rat1a-myc cells. These data demonstrate that the in vivo phenotype of RLR cells correlates well with the in vitro transformation phenotype. Together with our previous studies of Rcl and LDH-A in cell transformation, these data also suggest that Rcl and LDH-A are critical for the pathogenesis of tumors in which there is deregulated c-myc expression. In fact, elevated Rcl expression may be a component of the transformation program of several oncogenes. We have observed that rcl mRNA expression is strongly induced by oncogenic Ras in Rat1a fibroblasts and that rcl mRNA expression is elevated in all cancer cell lines in which we have analyzed its expression.5 Furthermore, we have previously demonstrated that elevated LDH-A expression, detected in many tumors, underlies the Warburg effect, the unique ability of tumor cells to undergo aerobic glycolysis (15) . This ability may contribute to the ability of tumor cells to proliferate under hypoxic conditions. Thus, while potentially very disparate in function, Rcl and LDH-A may represent potential targets for therapeutic intervention in tumors displaying aberrant c-myc expression.

To further analyze the role of Rcl in the induction of tumors in vivo, we asked whether Rcl can cooperate with VEGF, a protein found to be elevated frequently in human tumors (31) . Although not oncogenic itself, VEGF aids in tumor proliferation through the stimulation of angiogenesis and the consequently increased oxygen and nutrient supply (31) . Recent data suggest that, in addition to tumor cells, the stromal cells surrounding tumors may also be important producers of VEGF (32) . In experimental models, VEGF has been shown to be necessary the tumorigenicity of several oncogenes, including activated ras (33, 34, 35) .

Introduction of cells that simultaneously express Rcl and VEGF189 into nude mice demonstrated that VEGF could potentiate tumor induction by Rcl. Ninety-three percent of mice that received injections of cells from the Rcl-VEGF189 line developed tumors. These tumors were significantly larger than those induced by RLR cells and demonstrated a greater degree of vascularity in comparison with RLR tumors of approximately the same size. Thus, VEGF coexpression turns Rcl into a potent transforming molecule in vivo. These data support our hypothesis that deregulated Rcl expression allows inappropriate cell growth and proliferation, albeit by unknown mechanisms.

Despite their high vascularity, the Rcl-VEGF189 tumors displayed highly necrotic centers, which were infiltrated with eosinophils and neutrophils. Necrosis was not seen to a significant degree in tumors induced by Rat1a-myc or RLR cells. This suggests that elevated LDH-A expression, through either ectopic expression or induction by c-Myc, may protect the tumor cells in the hypoxic center of the tumor from cell death, and in this way contribute to tumor growth.

The data presented here represent the first direct evidence of cooperation between two c-Myc targets and confirm Rcl as an important downstream mediator of c-Myc function in tumor induction. Our findings also suggest that Rcl itself may stimulate tumor formation in the proper metabolic environment and can, therefore, be considered a potential new oncogene. Despite its apparent significance in cell transformation, the normal function of Rcl remains undetermined. Protein interaction screens have failed to identify any partner molecules.5 The protein bears no structural homology to known proteins, and examination of the yeast and worm genomes has failed to identify homologues in these organisms. The identification of a Drosophila homologue of c-myc suggests that a rcl homologue may exist in this organism (36 , 37) . Further elucidation of the in vivo function of Rcl, in mammalians or other species, will be important to understand the mechanisms of tumor induction by c-Myc and, potentially, other oncogenes.


   ACKNOWLEDGMENTS
 
We thank Jim Flook for expert assistance with flow cytometric analysis and members of our laboratory for insightful discussions and critical review of the manuscript.


   FOOTNOTES
 
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.

1 Supported by NIH Grant CA57341 (to C. V. D.). H. S. is a special fellow of the Leukemia Society of America. Back

2 Supported in part by a Howard Hughes predoctoral fellowship and a Lymphoma Research Foundation postdoctoral fellowship. Present address: Memorial Sloan Kettering Cancer Center, New York, NY 10021. Back

3 Supported by an American Society of Clinical Oncology Young Investigator Award. Present address: Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC 27599. Back

4 To whom requests for reprints should be addressed, at Ross Research Building, Room 1025, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205. Phone: (410) 955-2773; Fax: (410) 955-0185; E-mail: cvdang{at}welch.jhu.edu Back

5 B. C. Lewis and C. V. Dang, unpublished data. Back

6 http://www.ncbi.nlm.nih.gov/UniGene/. Back

7 The abbreviations used are: LDH-A, lactate dehydrogenase A; VEGF, vascular endothelial growth factor; RLR, Rat1a-LDH-rcl; RLP, Rat1a-LDH-pSG5; FBS, fetal bovine serum. Back

8 J. E. Prescott and C. V. Dang, unpublished data. Back

Received 12/13/99. Accepted 8/23/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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