Differential gene expression in a murine model of cancer cachexia

Constance L. Monitto1, Dan Berkowitz1, Kyoung Min Lee1, Sokhon Pin1, Daqing Li3, Michael Breslow1, Bert O'Malley3, and Martin Schiller1,2

Departments of 1 Anesthesiology and Critical Care Medicine and 2 Pathology, The Johns Hopkins Hospital, Baltimore, 21287; and 3 Department of Otorhinolaryngology and Head and Neck Surgery, University of Maryland Medical Center, Baltimore, Maryland 21201


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Murine adenocarcinoma 16 (MAC16) tumors and cell lines induce cachexia in NMRI nude mice, whereas histologically similar MAC13 tumors do not. After confirming these findings in BALB/c nude mice, we demonstrated that this tissue wasting was not related to decreased food intake or increased total body oxidative metabolism. Previous studies have suggested that MAC16's cachexigenic properties may involve the production of tumor-specific factors. We therefore screened for genes having increased expression in the MAC16 compared with the MAC13 cell line by performing hybridization to a murine cDNA expression array, by generation and comparison of cDNA libraries from each cell line, and by PCR-based subtractive hybridization. Northern blot hybridization was performed to confirm differences in transcript expression. Transcripts encoding insulin-like growth factor binding protein-4, cathepsin B, ferritin light and heavy chain, endogenous long-terminal repeat sequences, and a viral envelope glycoprotein demonstrated increased expression in the MAC16 cell line. The roles of a number of these genes in known metabolic pathways identify them as potential participants in the induction of cachexia.

murine adenocarcinoma 13 and 16 cells; cDNA expression array


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CACHEXIA, OR PROGRESSIVE TISSUE WASTING, is manifested by abnormalities in carbohydrate, lipid, and protein metabolism (34). Cachexia can occur in association with sepsis, trauma, and acquired immunodeficiency syndrome (4), as well as many types of cancer. It is one of the most important factors leading to early morbidity and mortality in cancer, accounting for 10-20% of all deaths, and contributing to deaths from other causes such as infection (11, 40). Despite its profound clinical impact, the mechanisms of cancer cachexia are still incompletely understood. Although anorexia may accompany cachexia, it does not appear to account for the skeletal muscle and adipose tissue wasting seen in cancer, since nutritional supplementation does not reverse the progressive weight loss observed (10), as occurs with refeeding of patients with anorexia nervosa (29). Cytokines, including tumor necrosis factor-alpha , interleukin (IL)-1, interferon-gamma , leukemia inhibitory factor, IL-6, and transforming growth factor-beta are thought to play a role in cancer cachexia (17, 23, 24, 26, 42, 43), but therapies aimed at suppressing the cytokine response have been only partially effective in preventing manifestations of the disease (8).

Implantable tumor models in mice provide a means to identify and screen potential candidate molecules important in the induction of cachexia. One such model utilizes two histologically similar colon cancer cell lines, murine adenocarcinoma (MAC)13 and MAC16, derived from tumors initially induced by administration of 1,2-dimethylhydrazine (6). Previous studies have demonstrated that NMRI mice implanted with MAC16 tumor cells develop a 20-30% loss in weight over the course of 1 mo after tumor cell implantation, whereas MAC13-implanted mice maintain their weight (19). The cachexigenic properties of MAC16 tumors do not appear to be cytokine mediated (25) but may involve the production and secretion of tumor-specific factors. Lipolytic factors appear to be produced by MAC16 and, to a lesser extent, MAC13 tumors (2). In addition, a 24-kDa glycopeptide isolated from MAC16 tumors and not present in MAC13 tumors has been shown to induce weight loss in vivo and skeletal muscle proteolysis in vitro (16). Moreover, serum from MAC16-implanted mice also increases muscle proteolysis in vivo (33). These findings suggest that differential production and/or secretion of factors produced by MAC16 tumor cells may play a role in the development of cancer cachexia by inducing lipolysis and proteolysis in peripheral tissues.

In this study, we confirmed the induction of cachexia by MAC16, but not MAC13, tumors in BALB/c athymic nude mice. Hypothesizing that this difference might be related to variations in gene expression between the two cell lines, we then utilized three different methods, 1) hybridization to a commercially available murine cDNA expression array, 2) generation and comparison of cDNA libraries from each cell line, and 3) PCR-based subtractive hybridization, to characterize differences in gene expression between MAC13 and MAC16 tumor cells. Partial profiles of gene expression patterns were generated for the two cell lines by these different approaches, and analysis of these expression patterns has led to the identification of several genes that are overexpressed in MAC16 tumor cells and may play a role in the etiology of cancer cachexia in this model.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor Cell Lines

MAC13 and MAC16 tumor cells were obtained from Dr. M. Tisdale (Pharmaceutical Sciences Institute, Aston University, Birmingham, UK) and were maintained in vitro in RPMI 1640 medium with L-glutamine (GIBCO BRL, Life Technologies, Rockville, MD) containing 12% fetal bovine serum (HyClone, Logan, UT) and penicillin-streptomycin (100 U/ml and 100 µg/ml, respectively) in a humidified atmosphere with 5% CO2 at 37°C. Cells were passaged once they achieved 80% confluency.

Mice

All animal procedures were approved by the Johns Hopkins University Institutional Animal Care and Use Committee. Six-week-old male athymic nude mice (BALB/c nu/nu) were obtained from the National Cancer Institute (Frederick, MD). Animals had access to standard laboratory rodent chow and water ad libitum throughout the course of the studies.

In Vivo Experiments

Tumor implantation. MAC13 and MAC16 tumor cells grown in culture to ~80% confluency were treated with trypsin, washed with medium, and resuspended at a concentration of 5 × 107 cells/ml in phosphate-buffered saline. A total of 100 µl of this cell suspension was injected subcutaneously into the thigh of each mouse.

Food intake, body weight, and metabolism. Food intake, body weight, and oxygen consumption (VO2) were measured every 3-4 days after tumor implantation. Total body VO2 was measured using indirect calorimetry with a four-chamber Oxymax system (Columbus Instruments, Columbus, OH), as previously described (3); the first 30 min of data per day were discarded to allow the mice to become acclimatized to their surroundings. VO2 per animal per day was calculated as the average VO2 over the time period measured. Upon completion of all metabolic measurements on postimplantation day 25, mice were euthanized with pentobarbital sodium intraperitoneally, and tumor tissue was excised and stored at -80°C.

In Vitro Techniques

RA isolation and Northern blot analysis. Total RNA was isolated from cell culture and tumor tissue by the RNA-STAT 60 reagent method (Tel-test "B," Friendswood, TX). MAC13 and MAC16 poly(A+) mRNA were prepared from total RNA with the use of the Promega PolyATract mRNA Isolation System IV kit (Promega, Madison, WI). Northern blot hybridization was performed as previously described (32). Briefly, total RNA samples (10 µg) were separated by electrophoresis on 1% agarose gels containing formaldehyde and then transferred onto nylon membranes by capillary action (Nytran, Schleicher and Schuell, Keene, NH). Blots were prehybridized for 1 h at 65°C in an SDS-PIPES hybridization buffer (32) and hybridized overnight with radiolabeled cDNA probes at 65°C. The membranes were washed three times at 65°C in 0.1× standard sodium citrate (SSC; 20× SSC is composed of 3 M sodium chloride and 0.3 M sodium citrate, pH 7.0) and 0.1% SDS and then exposed to X-ray film with intensifying screens at -80°C. Signals were normalized by probing of the identical blot with probes to beta -actin or S26 ribosomal protein cDNA. Signals were quantified using a phosphorimager and image analysis software (Imagequant, Molecular Dynamics, Sunnyvale, CA).

cDNA probes were generated from cDNA fragments by random priming with [alpha -32P]dCTP, as described by Stratagene (PrimeIT-II kit, Stratagene, La Jolla, CA). cDNA fragments encoding regions of cathepsin B (accession no. Y18463, nt 38-382), insulin-like growth factor binding protein-4 (IGFBP-4; accession no. X81582, nt 86-1028), and calgizzarin (accession no. AI317551, nt 103-503) were generated by polymerase chain reaction (PCR) from a MAC16 cDNA library. Clones encoding cDNAs for mouse ferritin light chain, ferritin heavy chain, mitochondrial uncoupling protein 2, peroxisomal acyl-CoA, and endo B cytokeratin (I.M.A.G.E. Consortium Clone ID nos. 1958025, 1970583, 19733292, 1973572, and 1973584, respectively) were purchased from American Tissue Culture Collection (ATCC; Manassas, VA). beta -Actin probe was purchased from Ambion (Austin, TX), and S26 probe was synthesized as previously described (37). Probes for sequences identified by subtractive hybridization were generated from cDNA fragments excised from individual pCR 2.1 constructs (see PCR-Based Subtractive Hybridization). Recombinant bacteria were then grown overnight in Luria Broth containing 100 µg/ml ampicillin. For preparation of all probes, plasmid DNAs were isolated by mini- or midiprep (QIAprep Spin Miniprep Kit and Plasmid Midi Kit, Qiagen, Valencia, CA), and fragments of interest were extracted from plasmids after restriction enzyme digestion (EcoRI and XhoI for ATCC clones and EcoRI for pCR 2.1 clones) and separation of DNA fragments on low-melting agarose gels. DNAs were purified from gel slices by gel extraction (QIAquick gel extraction kit, Qiagen).

Atlas cDNA Array Analysis

In accordance with the protocol provided by the manufacturer (Atlas cDNA Expression Array, Clontech Laboratories, Palo Alto, CA), radiolabeled cDNAs were synthesized from poly(A+) mRNA isolated from MAC13 and MAC16 tumor cells by use of a pool of 588 gene-specific primers. Individual hybridizations of the two different cDNA populations to similar Atlas cDNA arrays were performed, and hybridization intensities of the cDNAs on the arrays were compared. After hybridization, each membrane was washed and exposed to film. After films in which the intensities of the housekeeping genes were similar were obtained, films were scanned and overlaid to allow for identification of comparably expressed and differentially expressed genes.

Electronic Subtraction

Directional lambda ZAP2 cDNA libraries were generated from the MAC13 and MAC16 cell lines, and randomly selected clones were isolated and sequenced. The phage cDNA libraries were prepared as previously described (Lambda ZAP II Vector/Gigapack Cloning Kit, Stratagene). Briefly, poly(A+) RNA (6 µg) was hybridized to the primer 5'-GAGAGAGAGAGAGAGAGAGAAACTAGTCTGAGT(18)-3'. The RNA was reverse transcribed to generate the first strand of cDNA, and then second-strand cDNA was synthesized. The cDNAs were blunt ended and ligated to a phosphorylated EcoRI adapter. Next, the XhoI site within the oligo-dT primer was cut, the modified cDNAs were fractionated on a gel filtration column, and the larger-size cDNAs were pooled. Phage arms were ligated to the cDNAs, the resulting constructs were packaged using GigaPack III Gold, and library titers were determined. The average insert size was 1.7 kb, with cDNA sizes ranging from 0.4 to 12 kb. After mass excision of the MAC13 and MAC16 cDNA libraries, individual, randomly selected clones were arrayed at Lawrence-Livermore National Laboratory, and partial sequences were obtained from the 5' ends of randomly selected clones by means of the primer -40RP (GIBCO BRL, Life Technologies) and automated fluorescent-dye sequencing. The cDNAs were identified using basic local alignment search tool nucleotide (BLASTN; Ref. 1). Frequency of expression of each cDNA sequenced was quantitated, and cDNAs showing higher levels of expression in the MAC16 compared with the MAC13 library were identified.

PCR-Based Subtractive Hybridization

Suppression-subtractive hybridization, a PCR-based cDNA subtraction method, was used to selectively amplify differentially expressed cDNA fragments while simultaneously suppressing nontarget DNA amplification (7). In accordance with the protocol provided by the manufacturer (Clontech PCR-Select cDNA Subtraction Kit, Clontech Laboratories), mRNAs derived from MAC13 and MAC16 cells were converted into double-stranded cDNAs. Tester (MAC16) and driver (MAC13) cDNAs were hybridized, and the hybrid sequences were removed. The remaining molecules were then subjected to two rounds of PCR to amplify and enrich differentially expressed sequences. These cDNAs were inserted into the pCR 2.1 vector (Original TA Cloning Kit, Invitrogen, Carlsbad, CA) and transferred into Escherichia coli NM-522, and plasmids were prepared from eight randomly selected clones and analyzed by Northern blotting and DNA sequencing.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Weight Loss, Food Intake, and Metabolic Data

Previous work by others has demonstrated a 20% weight loss in male NRMI mice after MAC16 tumor cell implantation (6). We performed similar experiments to test this model in BALB/c athymic nude mice. As in NMRI mice, mice implanted with either MAC13 or MAC16 cells developed tumors, and significant weight loss was observed only in those mice implanted with MAC16 tumor cells (Fig. 1A). After implantation, animals bearing MAC16 tumors lost ~20% of body weight between days 14 and 25, whereas MAC13-implanted mice either maintained or gained weight, despite bearing a similar-size tumor mass. Body weight of MAC16-implanted mice was significantly less than that of MAC13-implanted mice by postimplantation day 18 (P < 0.05).


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Fig. 1.   Mice implanted with murine adenocarcinoma (MAC)16 tumors develop cachexia, whereas those implanted with MAC13 tumors do not. A: weight of MAC13 and MAC16 tumor-bearing mice (n = 7 per group). B: food intake of mice whose body weights are represented in A (n = 7 per group). C: total body oxygen consumption of MAC13- and MAC16-implanted mice (n = 4 per group). *P < 0.05.

To determine whether decreases in caloric intake or increases in total body oxidative metabolism could account for this weight loss, we measured food intake and VO2. Food intake did not significantly decrease until the mice had begun to lose weight by day 18 (P < 0.05) (Fig. 1B). In addition, total body VO2 (ml · kg-0.7 · h-1) did not increase in MAC16-implanted mice during this same time period; in fact, it decreased after the onset of weight loss (Fig. 1C). These findings suggest that neither a change in caloric intake nor total body oxidative metabolism was the immediate cause of the observed tissue wasting.

Identification of Upregulated Gene Transcripts

In light of previous studies suggesting that MAC16 tumors may induce cachexia by means of increased synthesis of lipolytic and proteolytic tumor gene products (2, 16, 36), we used three different approaches: hybridization to a cDNA expression array, comparison of cDNA libraries from each cell line, and PCR-based subtractive hybridization, to identify genes with elevated expression in MAC16 vs. MAC13 tumor cells. Differential expression of select genes identified by these preliminary screens was confirmed by Northern blot analysis. Because previous experiments had demonstrated the presence of putative cachexia-inducing factors in tumor tissue, serum, and possibly urine, we focused our attention on secretory products and genes with known metabolic impact in our secondary Northern blot screening.

Atlas cDNA array. Gene expression array analysis was performed to screen for differences in expression of 588 known mouse cDNAs (for full list of genes and array grid locations see http://www.clontech.com/atlas/genelists/index.html). We identified nine genes that showed different spot intensities upon comparison of MAC13 and MAC16 probed arrays (Fig. 2 and Table 1). Two of these genes, RNA polymerase I termination factor and murine ornithine decarboxylase, had increased intensity in the MAC13 cell line, whereas the remaining genes were higher in the MAC16 cell line. Although attention has been focused in other cancer cachexia models on the role of IL-6 and other cytokines as potential contributors to cachexia, expression of IL-6 was not increased in MAC16 compared with MAC13 cells by either array or Northern blot analysis (data not shown). Northern blot analysis of tumor-derived RNA and quantitation of signals using a phosphorimager revealed more than a twofold increase in expression of IGFBP-4, and more than a fourfold increase in expression of cathepsin B in MAC16 compared with MAC13 total RNA (Fig. 3).


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Fig. 2.   Atlas cDNA hybridization arrays for MAC13 and MAC16 mRNA. Radiolabeled cDNA was synthesized from poly(A+) mRNA (1 µg) isolated from MAC13 and MAC16 tumor cells by use of a pool of 588 gene-specific primers. Individual hybridizations of the 2 different RNA populations were performed and membranes were washed and exposed to film. After films were obtained in which the intensity of the housekeeping genes was similar, they were scanned and overlaid to allow for identification of comparably and differentially expressed genes. Genes that appeared significantly different upon comparison of MAC13 and MAC16 probed arrays are denoted by a  and identified by grid location in Table 1.


                              
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Table 1.   Genes with differential expression between MAC13 and MAC16 cell lines as determined by cDNA hybridization array



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Fig. 3.   Northern blot analysis of select genes with increased expression in MAC16 tumors identified by Atlas cDNA array. Each lane contains tumor RNA (10 µg). All signals were normalized to S26. Nos. 13-1, -2, -3, -4 and 16-1, -2, -3, -4 refer to individual MAC13 and MAC16 tumors, respectively. Insulin-like growth factor binding protein (IGFBP)-4 (2.6 kb) and cathepsin B (2.2 kb) messages were present at elevated levels in MAC16, compared with MAC13, tumors (P < 0.05).

Electronic subtraction. cDNA libraries were generated for the MAC13 and MAC16 cell lines. Randomly selected clones were arrayed, and partial sequences were obtained from the 5' ends of 231 MAC16 and 649 MAC13 clones (for full list see Entrez Browser nucleotide database, with Schiller and MAC13 or MAC16 as queries). These clones included 489 sequences present in the nonredundant database, 129 sequences present in the expressed sequence tag (EST) database, and 29 unique sequences. Frequency of clone expression was tabulated, and a normalized ratio was calculated on the basis of the relative contribution of MAC16 and MAC13 clones to the total number of clones sequenced (1:2.8).

Although electronic subtraction provides digital data, it is most useful in identifying abundant mRNAs (39). Hence, given the limited size of our total database (880 clones) and our inability to differentiate with any degree of certainty between the probable frequency of sequences expressed between zero and one time in each library, we focused our attention on more frequently expressed transcripts and excluded all single transcripts (MAC13: 405 sequences; MAC16: 146 sequences) from further study. The electronic subtraction data suggested that 16 transcripts that occurred at least two times in the MAC16 library might have increased expression in MAC16 compared with MAC13 cells (Table 2). Six of these transcripts, including a known secretory protein (ferritin), as well as a protein capable of influencing cellular metabolism (mitochondrial uncoupling protein 2), were selected for secondary screening by Northern blot analysis. Using this approach we confirmed a four- to fivefold increase in expression of both ferritin heavy-chain and ferritin light-chain mRNA in MAC16 cell culture and tumor tissue compared with MAC13 cell culture and tumor tissue (Fig. 4). In contrast to our electronic subtraction data, we observed comparable expression levels of endo B cytokeratin and mitochondrial uncoupling protein 2, and increased expression of the calcium binding protein calgizzarin (35) was observed in MAC13 cell culture-derived RNA. We were unable to detect expression of peroxisomal acyl-CoA oxidase message by Northern blot analysis for either cell line with our probe. With examination of the 29 novel sequences, each was found to occur only once in the combined library database, suggesting that this technique was not helpful in identifying differential expression of novel clones.

                              
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Table 2.   Sequences demonstrating differential expression between MAC16 and MAC13 cells, as determined by electronic subtraction of cDNA libraries



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Fig. 4.   Northern blot analysis of ferritin light- chain and ferritin heavy-chain expression. Each lane contains tumor RNA (10 µg). All signals were normalized to S26. Ferritin light chain (0.8 kb) and heavy-chain (1 kb) messages are present at >4-fold higher levels in MAC16 cells (P < 0.05).

PCR-select subtraction. To screen more extensively for potential novel and/or rare transcripts differentially expressed in the MAC cell lines, PCR-select subtraction was performed. Eight of the resulting cDNAs were partially sequenced, and expression levels were compared by Northern blot analysis. By comparison of these partial sequences to the GenBank database, five clones were initially found to demonstrate homology to previously identified genes, whereas two (clones 6 and 18) were homologous to sequences reported in the EST database (Table 3). One additional novel clone, clone 10, was subsequently used to further probe a MAC16 cDNA library, and a larger clone was identified and sequenced and was found to have high homology to the 3' end of a retroviral envelope protein sequence (data not shown). Northern blot analysis revealed no difference in expression of clone 7 (calcium-binding protein ALG-2) in MAC16 vs. MAC13 cells, less than a twofold increase in expression of clone 1 (human putative enterocyte differentiation-promoting factor) in MAC16 vs. MAC13 tumor-derived RNA, and two- to threefold increases in expression of clones 5 (goblet cell mucus-secreting protein GOB-4), and 6 (AI482147) in MAC16 cells. Clones 10 (retroviral envelope glycoprotein), 12 (mouse endogenous retrovirus 3' long-terminal repeat), and 18 (AA395982) were highly expressed by MAC16 cells but demonstrated barely detectable expression in MAC13 cells, and this finding was confirmed in tumor-derived RNA for clones 10 and 18 (Fig. 5). These data indicate that PCR select-subtractive hybridization is useful in identifying previously unreported novel and known differentially expressed genes in this cancer cachexia model.

                              
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Table 3.   cDNAs having increased expression in MAC16 cells, as identified by PCR-select hybridization



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Fig. 5.   Northern blot analysis of clones 10 (envelope glycoprotein) and 18 (EST AA395982), identified by PCR-select subtraction. Each lane contains tumor RNA (10 µg). All signals were normalized to beta -actin or S26. Transcripts for clones 10 and 18 were detectable in MAC16, but not MAC13, tumors.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Implantable tumors in animals provide a model to further elucidate the cellular mechanisms involved in cancer cachexia. In the present study, we focused on two murine adenocarcinoma cell lines with readily observable differences in their ability to induce cachexia in mouse models. Despite generation of these cell lines by similar treatment and their similar histological appearance, MAC13 and MAC16 tumors are quite distinct in their ability to induce weight loss. As previously demonstrated in NMRI mice, we confirmed that MAC16 tumor cell implantation and growth induced substantial weight loss in BALB/c nude mice. On the basis of our metabolic and food intake data, this wasting does not appear to be related to alterations in caloric intake, as may occur with tumor necrosis factor-induced anorexia (21) or to differences in total body VO2. In addition, weight loss induced by MAC16 tumors was neither strain specific nor dependent on host T-cell-mediated immunity.

Given the identical origin of these two cell lines, it is likely that their genetic profiles are not vastly different. We hypothesized that differences in a limited number of genetic events and/or gene expression may reveal differences that underlie important phenotypic characteristics, such as the ability to induce cachexia. To screen and compare the genetic expression profiles of these two cell lines, we utilized three different but complementary approaches. Although each technique was limited and did not allow a comprehensive screening of all genes expressed by the two cell lines, each approach identified additional genes that were subsequently confirmed to be differentially expressed in these cell lines. In this manner, we were able to identify several genes and ESTs having higher levels of expression in MAC16 compared with MAC13 tumor cells.

A number of the genes identified by this screen are known to be upregulated in cancer. IGFBP-4 (20), cathepsin B (31), ferritin (22), endogenous long-terminal repeat sequences (28), and envelope protein-related transcripts (9) have previously been shown to be expressed in colon cancer cell lines. However, this is the first study to report differential expression of these genes between two adenocarcinoma cell lines with differing ability to induce cachexia. Of note, at least four of the genes identified by this screen are secretory proteins (IGFBP-4, ferritin light chain, ferritin heavy chain, and GOB-4). In addition, the normally intracellular protein cathepsin B has been demonstrated to be released from adenocarcinomas (15, 27). This is particularly relevant because previous work demonstrating cachexia-inducing effects of serum from MAC16-implanted mice, as well as isolation of proteolysis- and lipolysis-inducing factors, suggests that secretory products produced by this tumor may be important in cachexia.

Because this study screened for differences in gene expression between only two cell lines, the general role of this differential expression in the overall development of cachexia is unknown. However, the biological attributes of a number of these genes identify them as potential participants in the induction of cachexia. IGFBP-4 binds to IGF, inhibiting its interaction with the IGF receptor (12), and overproduction of IGFBP-4 could induce cachexia by preventing IGF-mediated anabolic effects in muscle and adipose tissue (18). In addition, IGFBP-4 might enhance muscle proteolysis by preventing IGF-mediated degradation of an mRNA transcript encoding the 14-kDa ubiquitin-conjugating enzyme E2, which catalyzes the first, irreversible step in the ubiquitin-dependent proteolytic system (41). Increased expression of the lysosomal protease cathepsin B might contribute to cachexia by inducing proteolysis by nonubiquitin-dependent pathways locally within the tumor. In addition, if released from tumor cells, cathepsin B might induce abnormal proteolysis of extracellular factors and tissues. The increase in both ferritin light- and ferritin heavy-chain mRNA in MAC16 tumor cells suggests that these cells may have an enhanced requirement for iron. By acting as an iron sink, the tumor might promote peripheral tissue degradation to provide a source of additional iron. Finally, increases in viral envelope glycoprotein or an endogenous long-terminal repeat sequence might promote cachexia by inducing the production of cytokines or arachidonic acid metabolites (38) or by promoting the transcriptional activation (5) of other intracellular cachexia-causing genes not identified by our screens.

A number of other sequences identified have homology to transcripts for proteins that have a less-established role in pathways that may be relevant to cachexia. For example, human putative enterocyte differentiation-promoting factor is a ubiquitin-conjugating E2 enzyme variant that lacks enzymatic activity but may be involved in the control of differentiation by altering cell cycle distribution (30). GOB-4 most likely generates a secretory protein involved in mucus-secreting function, in keeping with the gastrointestinal origin of these tumors (13), whereas pendulin (clone 11) acts as a blood cell tumor suppressor and is required for normal growth regulation (14). Variation of expression levels of these genes appears more likely to be related to the degree of differentiation of these tumor cell lines and may not be associated with modulation of adipose tissue or protein catabolism. Finally, clone 18 demonstrates barely detectable expression in MAC13 and high expression in MAC16 cell lines, but a potential role for this gene awaits further identification of its full-length sequence and characterization of its protein product.

There are a number of limitations to this study. Our approach provides a sampling, but not a comprehensive screening, of differences in gene expression between these two cell lines. Also, in our screen, we focused our attention on genes for secretory peptides with increased expression in MAC16 tumor cells. Hence, we may have failed to identify important factors because they were not identified by our screen or because they were related to decreased expression of genes in MAC16 cells or increased expression of genes in MAC13 cells. Moreover, clinically important effects may be the result of small changes in expression of critically important genes, whereas we focused on genes demonstrating more substantial differences in expression. A further limitation is the restriction of our screen to changes in mRNA expression, which provides little insight into differences induced by posttranslational modification of proteins.

Screening of tumor cell line gene expression profiles between a cachexigenic and a similar, noncachexigenic cell line identified differences in expression of genes important in known metabolic pathways. Further study is required to determine whether these observed changes translate into changes in protein production and/or activity to better characterize their contribution to the cachectic phenotype. Reproduction of wasting in mice by one or more of these protein products will further support their role in cancer-induced cachexia.


    ACKNOWLEDGEMENTS

The authors thank Dr. Michael Tisdale for provision of MAC13 and MAC16 cells, David Palmer for technical assistance, and Drs. Michael Wang and David Sidransky for advice.


    FOOTNOTES

C. L. Monitto is a Foundation for Anesthesia Education and Research/Baxter Pharmaceutical Research Starter Grant Recipient. This work was performed by the Department of Anesthesiology and Critical Care Medicine at the Johns Hopkins Hospital, Baltimore, MD.

Address for reprint requests and other correspondence: C. L. Monitto, Dept. of Anesthesiology/Critical Care Medicine, The Johns Hopkins Hospital, 600 North Wolfe St./Blalock 943, Baltimore, MD 21287 (E-mail: cmonitto{at}welchlink.welch.jhu.edu).

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

Received 13 October 2000; accepted in final form 6 March 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Altschul, SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, and Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389-3402, 1997[Abstract/Free Full Text].

2.   Beck, SA, Mulligan HD, and Tisdale MJ. Lipolytic factors associated with murine and human cancer cachexia. J Natl Cancer Inst 82: 1922-1926, 1990[Abstract].

3.   Breslow, MJ, Min-Lee K, Brown DR, Chacko VP, Palmer D, and Berkowitz DE. Effect of leptin deficiency on metabolic rate in ob/ob mice. Am J Physiol Endocrinol Metab 276: E443-E449, 1999[Abstract/Free Full Text].

4.   Bruera, E. Current pharmacological management of anorexia in cancer patients. Oncology 6: 125-130, 1992[Medline].

5.   Choi, SY, and Faller DV. The long terminal repeats of a murine retrovirus encode a trans-activator for cellular genes. J Biol Chem 269: 19691-19694, 1994[Abstract/Free Full Text].

6.   Cowen, DM, Double JA, and Cowen PN. Some biological characteristics of transplantable lines of mouse adenocarcinomas of the colon. J Natl Cancer Inst 64: 675-681, 1980[ISI][Medline].

7.   Diatchenko, L, Lau YC, Campbell AP, Chenchick A, Moqadam F, Huang B, Lukyanoz S, Lukyanov K, Gurskaya N, Sverdlov ED, and Siebert PD. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 93: 6025-6030, 1996[Abstract/Free Full Text].

8.   Fujimoto-Ouchi, K, Tamura S, Mori K, Tanaka Y, and Ishitsuka H. Establishment and characterization of cachexia-inducing and -non-inducing clones of murine colon 26 carcinoma. Int J Cancer 61: 522-528, 1995[ISI][Medline].

9.   Gottoni-Celli, S, Kirsch K, Kalled S, and Isselbacher KJ. Expression of type C-related endogenous retroviral sequences in human colon tumors and colon cancer cell lines. PNAS USA 83: 6127-6131, 1986[Abstract].

10.   Heber, D, Byerly LO, Chi J, Grosvenor M, Bergman RN, Coleman M, and Chlebowski RT. Pathophysiology of malnutrition in the adult cancer patient. Cancer 58: 1867-1873, 1986[ISI][Medline].

11.   Inagalo, K, Rodriguez V, and Bodey GP. Proceedings: causes of death in cancer patients. Cancer 33: 568-573, 1974[ISI][Medline].

12.   Jones, JI, and Clemmons DR. Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16: 3-34, 1995[ISI][Medline].

13.   Komiya, T, Tanigawa Y, and Hirohashi S. Cloning of the gene gob-4, which is expressed in intestinal goblet cells in mice. Biochim Biophys Acta 1444: 434-438, 1999[ISI][Medline].

14.   Kussel, P, and Frasch M. Pendulin, a Drosophila protein with cell cycle-dependent nuclear localization, is required for normal cell proliferation. J Cell Biol 129: 1491-1507, 1995[Abstract].

15.   Linebaugh, BE, Sameni M, Day NA, Sloane BF, and Keppler D. Exocytosis of active cathepsin B enzyme activity at pH 7.0, inhibition and molecular mass. Eur J Biochem 264: 100-109, 1999[Abstract/Free Full Text].

16.   Lorite, MJ, Cariuk P, and Tisdale MJ. Induction of muscle protein degradation by a tumour factor. Br J Cancer 76: 1035-1040, 1997[ISI][Medline].

17.   Matthys, P, Dijkmans R, Proost P, Van Damme J, Heremans H, Sobis H, and Billiau A. Severe cachexia in mice inoculated with interferon-gamma -producing tumor cells. Int J Cancer 49: 77-82, 1991[ISI][Medline].

18.   Mauras, N, and Haymond MW. Metabolic effects of recombinant human insulin-like growth factor-I in humans: comparison with recombinant human growth hormone. Pediatr Nephrol 10: 318-323, 1996[ISI][Medline].

19.   McDevitt, TM, and Tisdale MJ. Tumour-associated hypoglycaemia in a murine cachexia model. Br J Cancer 66: 815-820, 1992[ISI][Medline].

20.   Michell, NP, Langman MJ, and Eggo MC. Insulin-like growth factors and their binding proteins in human colonocytes: preferential degradation of insulin-like growth factor binding protein 2 in colonic cancers. Br J Cancer 76: 60-66, 1997[ISI][Medline].

21.   Michie, HR, Sherman ML, Spriggs DR, Rounds J, Christie M, and Wilmore DW. Chronic TNF infusion causes anorexia but not accelerated nitrogen loss. Ann Surg 209: 19-24, 1989[ISI][Medline].

22.   Modjtahedi, N, Frebourg T, Fossar N, Lavialle C, Cremisi C, and Brison O. Increased expression of cytokeratin and ferritin-H gene in tumorigenic clones of the SW 613-S human colon carcinoma cell line. Exp Cell Res 201: 74-82, 1992[ISI][Medline].

23.   Moldawer, LL, Georgieff M, and Lundholm K. Interleukin 1, tumor necrosis factor-alpha (cachectin) and the pathogenesis of cancer cachexia. Clin Physiol 7: 263-274, 1987[ISI][Medline].

24.   Mori, M, Yamaguchi K, Honda S, Nagasaki K, Ueda M, Abe O, and Abe K. Cancer cachexia syndrome developed in nude mice bearing melanoma cells producing leukemia-inhibitory factor. Cancer Res 51: 6656-6659, 1991[Abstract].

25.   Mulligan, HD, Mahony SM, Ross JA, and Tisdale MJ. Weight loss in a murine cachexia model is not associated with the cytokines tumor necrosis factor-alpha or interleukin-6. Cancer Lett 65: 239-243, 1992[ISI][Medline].

26.   Oliff, A, Defeo-Jones D, Boyer M, Martinez D, Kiefer D, Vuocolo G, Wolfe A, and Socher SH. Tumor secreting human TNF/cachectin induce cachexia in mice. Cell 50: 555-563, 1987[ISI][Medline].

27.   Recklies, AD, Tiltman KJ, Stoker TA, and Poole AR. Secretion of proteinases from malignant and nonmalignant human breast tissue. Cancer Res 40: 550-556, 1980[Abstract].

28.   Royston, ME, and Augenlicht LH. Biotinated probe containing a long-terminal repeat hybridized to a mouse colon tumor and normal tissue. Science 23: 1339-1341, 1983.

29.   Salisbury, JJ, Levine AS, Crow SJ, and Mitchell JE. Refeeding, metabolic rate, and weight gain in anorexia nervosa: a review. Int J Eat Disord 17: 337-345, 1995[ISI][Medline].

30.   Sancho, E, Vila MR, Sanchez-Pulido L, Lozano JJ, Paciucci R, Nadal M, Fox M, Harvey C, Bercovich B, Loukili N, Ciechanover A, Lin SL, Sanz F, Estivill X, Valencia A, and Thomson TM. Role of UEV-1, an inactive variant of the E2 ubiquitin-conjugating enzymes, in in vitro differentiation and cell cycle behavior of HT-29-M6 intestinal mucosecretory cells. Mol Cell Biol 18: 576-589, 1998[Abstract/Free Full Text].

31.   Satoh, Y, Higashi T, Nouso K, Shiota T, Kinugasa N, Yoshida K, Uematsu S, Nakatsukasa H, Nishimura Y, and Tsuji T. Cathepsin B in the growth of colorectal cancer: increased activity of cathepsin B in human colorectal cancer. Acta Med Okayama 50: 305-311, 1996[ISI][Medline].

32.   Schiller, MR, and Darlington DN. Stage-specific expression of RESP18 in the testes. J Histochem Cytochem 44: 1489-1496, 1996[Abstract].

33.   Smith, KL, and Tisdale MJ. Mechanism of muscle protein degradation in cancer cachexia. Br J Cancer 68: 314-318, 1993[ISI][Medline].

34.   Tisdale, MJ. Biology of cachexia. J Natl Cancer Inst 89: 1763-1773, 1997[Abstract/Free Full Text].

35.   Todoroki, H, Kobayashi R, Watanabe M, Minami H, and Hidaka H. Purification, characterization, and partial sequence analysis of EF-hand type 13-kDa Ca(2+)-binding protein from smooth muscle tissues. J Biol Chem 266: 18668-18673, 1991[Abstract/Free Full Text].

36.   Todorov, P, Cariuk P, McDevitt T, Coles B, Fearon K, and Tisdale M. Characterization of a cancer cachectic factor. Nature 379: 739-742, 1996[ISI][Medline].

37.   Vincent, S, Marty L, and Fort P. S26 ribosomal protein RNA: an invariant control for gene regulation experiments in eukaryotic cells and tissues. Nucleic Acids Res 21: 1498, 1993[ISI][Medline].

38.   Wahl, LM, Corcoran ML, Pyle SW, Arthru LO, Harel-Bellan A, and Farrar WL. Human immunodeficiency virus glycoprotein (gp120) induction of monocyte arachidonic acid metabolites and interleukin 1. PNAS USA 86: 621-625, 1989[Abstract].

39.   Wan, JS, Sharp SJ, Poirier GM, Wagaman PC, Chambers J, Pyati J, Hom YL, Galindo JE, Huvar A, Peterson PA, Jackson MR, and Erlander MG. Cloning differentially expressed mRNAs. Nat Biotechnol 14: 1685-1691, 1996[ISI][Medline].

40.   Warren, S. The immediate causes of death in cancer. Am J Med Sci 184: 610-615, 1932.

41.   Wing, SS, and Bedard N. Insulin-like growth factor I stimulates degradation of an mRNA transcript encoding the 14 kDa ubiquitin-conjugating enzyme. Biochem J 319: 455-461, 1996[ISI][Medline].

42.   Yoneda, T, Nakai M, Moriyama K, Scott L, Ida N, Kunitomo T, and Mundy GR. Neutralizing antibodies to human interleukin 6 reverse hypercalcemia associated with a human squamous carcinoma. Cancer Res 53: 737-740, 1993[Abstract].

43.   Zugmaier, G, Paik S, Wilding G, Knabbe C, Bano M, Lupu R, Deschauer B, Simpson S, Dickson RB, and Lippman M. Transforming growth factor beta 1 induces cachexia and systemic fibrosis without an antitumor effect in nude mice. Cancer Res 51: 3590-3594, 1991[Abstract].


Am J Physiol Endocrinol Metab 281(2):E289-E297
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society




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