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(American Journal of Pathology. 2000;156:1937-1950.)
© 2000 American Society for Investigative Pathology

Regular Articles

In Vivo Expression of the Novel CXC Chemokine BRAK in Normal and Cancerous Human Tissue

Mitchell J. Frederick^*, Ying Henderson^*, Xiaochun Xu, Michael T. Deavers, Aysegul A. Sahin, Hong Wu, Dorothy E. Lewis^§, Adel K. El-Naggar and Gary L. Clayman^*¶

From the Departments of Head and Neck Surgery,^*
Clinical Cancer Prevention,
Pathology,
and Cancer Biology,^¶
The University of Texas M. D. Anderson Cancer Center; and the Department of Immunology,^§
Baylor College of Medicine, Houston, Texas

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Note Added in Proof References

 
Using differential display, we cloned a gene with reduced expression in short-term explants of head and neck squamous cell carcinoma (HNSCC) tumors compared to cultured normal oral epithelial cells. The differentially expressed gene was identical to the recently cloned CXC chemokine BRAK, which is ubiquitously expressed in normal tissue extracts but is absent from many tumor cell lines in vitro. To define the cell populations expressing BRAK in vivo, in situ mRNA hybridization was performed on normal and cancerous tissues from six different histological sites. The predominant normal cell type constitutively expressing BRAK in vivo was squamous epithelium. Expression in tumors was heterogeneous, with the majority of HNSCCs and some cervical squamous cell carcinomas (SCCs) showing loss of BRAK mRNA. Although absent in unstimulated peripheral blood mononuclear cells, high levels of BRAK were consistently found in infiltrating inflammatory cells (with lymphocyte morphology) in nearly all cancers examined. Furthermore, BRAK expression was demonstrated in B cells and monocytes, after stimulation of peripheral blood mononuclear cells with lipopolysaccharide. This study demonstrates for the first time up-regulation of BRAK mRNA by inflammatory cells in the tumor microenvironment and lost expression from certain cancers in vivo. The data suggest that BRAK may have a role in host-tumor interactions.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Note Added in Proof References

Chemokines are a superfamily of small cytokines that selectively attract and activate leukocytes and a variety of other cell types.² Although their primary function appears to be directing leukocyte migration and endothelial transmigration, it is now clear that these small cytokines play a role in a variety of homeostatic and disease processes, including development, hematopoiesis, allergies, inflammatory syndromes, angiogenesis, and cancer.^2-5 The majority of chemokines are expressed in response to some stimuli, but several are constitutively produced.^2,4,5 In this study, we characterize the full-length cDNA encoding BRAK, examine by in situ hybridization the cell populations expressing this novel chemokine in normal and cancerous tissues from six different histological sites, and define populations of leukocytes capable of expressing BRAK.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Note Added in Proof References

 
Explants and Tumor Cell Lines

Explantation of human normal oral epithelial cells and HNSCC was performed using a protocol established by Dr. Peter Sacks (Memorial Sloan-Kettering Hospital, New York). Surgically removed specimens were histopathologically verified, minced, placed epithelial side up (for normal), and allowed to adhere and grow in AmnioMAX medium (Gibco BRL, Grand Island, NY). At first passage, contaminating fibroblasts were removed with cold trypsin, and remaining normal oral epithelial cells or HNSCC recovered by further trypsinization were seeded into 10-cm plates containing KGM growth medium (Clonetics, San Diego, CA). Tu-177, Tu-182, Tu-212, Tu-159, Tu-167, Tu-138, and MDA 1483 are established human HNSCC lines derived from larynx, tonsil, larynx, tongue, floor of the mouth, lip, and retromolar trigone, respectively, and have previously been characterized.^6;7 All tumor lines were passaged in Dulbecco’s minimum essential medium containing 10% fetal bovine serum (FBS), glutamine, penicillin, and streptomycin.

DDRT-PCR

Total RNA was isolated from explants with the TRI reagent (Molecular Research, Cincinnati, OH), and contaminating DNA was removed by DNase I treatment. DDRT-PCR was performed using a kit (Display Systems, Los Angeles, CA) that employs 24 arbitrary upstream primers and nine downstream primers (dT11VN). PCR products labeled with [-³³P]dATP were resolved by nondenaturing polyacrylamide gels and excised following autoradiography. Recovered fragments were reamplified, cloned into the pCR-TRAP plasmid (GeneHunter, Nashville, TN), and sequenced.

Nucleic Acid Searches and Sequence Alignments

The BLASTN and BLASTP searches were performed using the National Center for Biotechnology Information website (http://www.ncbi.nlm.nig.gov/), and open reading frames were identified using the ExPasy website translation tool software (http://expasy.hcuge.ch/www/dna.html). Multiple protein sequence alignments and subsequent phylogenetic analysis were performed using the ClustalW Multiple Sequence Alignment Program (at http://www2.ebi.ac.uk/clustalw/). Pairwise sequence alignment between BRAK cDNA and the BAC clone was accomplished with the LALIGN program at Genestream (http://vega.crbm.cnrs-mop.fr/bin/lalign-guess.cgi), and exon/intron boundaries were confirmed using the HSPL program available at the Baylor College of Medicine Search Launcher (http://kiwi.imgen.bcm.tmc.edu:8088/search-launcher/launcher.html).

Northern Blots

Total RNA (7.5 µg/sample) isolated from cell lines or biopsy specimens was fractionated by agarose-formaldehyde gel electrophoresis, transferred to a Hybond-N+ membrane (Amersham, Arlington Heights, IL), and hybridized with a -³²P-labeled 213-bp probe complementary to the entire coding region of BRAK. The same BRAK probe was also hybridized to a multitissue poly A⁺ RNA blot purchased from Clontech (Palo Alto, CA).

In Situ mRNA Hybridization

A total of 52 archival formalin-fixed paraffin-embedded specimens containing cancerous and/or adjacent normal tissue were obtained from 44 patients diagnosed with cancer of the head and neck, colon, kidney, cervix, breast, or ovary. In addition, one of the normal cervical samples was taken from a healthy volunteer who did not have cancer. In situ sense and antisense probe templates were prepared by amplifying a 367-bp fragment of BRAK cDNA (nucleotides 474–840), using sense and antisense primers modified with either T7 or SP6 sequences. The resulting templates were then transcribed with a digoxigenin RNA labeling kit (Boehringer Mannheim, Indianapolis, IN). After deparaffinization, proteinase K treatment, and hybridization, digoxigenin riboprobes were detected with the Genius 3 Nucleic acid kit (Boehringer Mannheim), which contains an anti-digoxigenin alkaline phosphatase-conjugated antibody and chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT), which appears as a purplish-brown precipitate. All slides were evaluated by two investigators, including a clinical pathologist. Tumor or corresponding normal tissue was scored + when 10% total cells present showed staining, or cells from more than one microscopic field under a 10x objective lens contained areas with 10% cells staining positive. Tumor or normal tissue was scored ± when only a single microscopic field under a 10x objective contained 10% cells staining positive on an entire slide. Staining was qualified as weak when the intensity of positively staining cells was less than 50% of that observed with normal squamous epithelium of the tongue. Inflammatory and stromal cells were considered + when multiple fields under a 10x objective contained positively staining cells with a signal intensity similar to that of normal squamous epithelium of the tongue, and as "weak+" when multiple fields contained positively staining cells with an intensity less than 50% of that observed with normal squamous epithelium of the tongue. All tissues failing to meet at least one of the above criteria were considered negative (-).

Reverse Transcription-Based PCR

RNA was isolated from cells using TRI reagent according to suppliers’ instructions, except that glycogen was added as a carrier molecule. Up to 2 µg total RNA was reverse-transcribed in a final reaction volume of 25 µl containing 200 ng random hexamers (PE Applied Biosystems, Foster City, CA), 1x first stand synthesis buffer (Gibco BRL), 0.4 mmol/L each of the deoxynucleoside triphosphates, 10 mmol/L dithiothreitol, 1 µl RNase inhibitor (Boehringer Mannheim), and 200 U SuperScript II reverse transcriptase (Gibco BRL). For sorted cells (ie, <3 x 10⁶ total), the entire RNA yield was reverse-transcribed in 25 µl. After cDNA synthesis, residual RNA was removed by digestion with 1 U RNase H (Boehringer Mannheim) at 37°C for 20 minutes. For PCR, 4 µl of cDNA reaction was amplified in a final volume of 30 µl containing 1x Mg²⁺-free Taq buffer, 50 µmol/L each deoxynucleoside triphosphate, 1.5 mmol/L Mg²⁺, 0.5 µmol/L gene-specific primers, and 1.5 U Promega Taq enzyme. PCR amplification was performed at 94°C for 30 seconds, followed by 35 cycles of 94°C for 30 seconds, 60°C for 20 seconds, 72°C for 50 seconds, and a final extension at 72°C for 7 minutes. PCR products were resolved by agarose electrophoresis and stained with ethidium bromide. BRAK-specific primers (derived from exons II and IV) amplifying a 233-bp amplicon were 5'-GTCCAAATGCAAGTGCTCCC-3' (sense) and 5'-TTCTTCGTAGACCCTGCGCT-3' (antisense). Primers (derived from sequences in exons II, III, and IV) specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)amplifyinga 231-bp fragment were 5'-ACGGATTTGGTCGTATTG-3' (sense) and 5'-TGATTTTGGAGGGATCTC-3' (antisense).

Peripheral Blood Mononuclear Cells and Cell Sorting

Heparinized whole blood obtained from healthy volunteers by venipuncture was separated over Histopaque 1077 (Sigma, St. Louis, MO) to obtain peripheral blood mononuclear cells (PBMCs). PBMCs were cultured in RPMI containing 10% FBS, with glutamine, penicillin, and streptomycin additives before stimulations. Phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187, and lipopolysaccharide (LPS) were purchased from Sigma. Phytohemagglutinin (PHA) and pokeweed mitogen (PWM) were from GibcoBRL, and recombinant human interferon- (rhIFN-) was from R&D (Minneapolis, MN). Recombinant human tumor necrosis factor (TNF) and interleukin-2 (IL-2) were generous gifts from Dr. Elizabeth Grimm (University of Texas M. D. Anderson Cancer Center, Houston, TX). PBMCs were stimulated for 6–8 hours with PMA (10 ng/ml), calcium ionophore (0.5 µM), PHA (1.5% final concentration), PWM (1.5% final concentration), LPS (0.5 µg/ml), TNF (500 U/ml), IFN- (200 U/ml), or IL-2 (500 U/ml). Subsequent to stimulation, nonadherent and adherent cells (harvested by EDTA treatment) were pooled and either used directly for RNA isolation or stained with antibodies for cell sorting. Phycoerythrin-cyanine 5-conjugated CD33, phycoerythrin-conjugated CD3, and fluorescein isothiocyanate-conjugated CD19 recognizing cell surface markers on monocytes, T cells, and B cells, respectively, were purchased from Beckman Coulter (Miami, FL). Cells were sorted, using a high-pressure Beckman Coulter ALTRA flow cytometer, into subpopulations that were >95% pure.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Note Added in Proof References

 
Isolation of BRAK cDNA

Short-term primary explants were established using tumor and normal biopsies from a patient with invasive SCC of the tongue, and extracted mRNA was used for DDRT-PCR. One of the gene fragments identified, N0402A (Figure 1A) , was expressed at much lower levels in the tumor explants (T1) compared to normal (noncancerous) oral epithelial explants (N1). After the 120-bp gene fragment N0402A was cloned and sequenced, a BLASTN search identified more than 95% homology with an EST (W73085), which was subsequently matched to the tentative human consensus (THC) sequence THC221548 in the TIGR Human Gene Index database. Although THC221548 contained 1105 bp of sequence, no long open reading frames (ORFs) were apparent. A series of successive BLASTN searches using end sequence information linked THC221548 to a set of homologous ESTs containing an ORF. Because the translation initiation start site was missing from the ESTs with the ORF, 5' rapid amplification of cDNA ends (RACE) was used to define upstream nucleotides. The cDNA including the translation start site, remaining ORF, and 3' untranslated sequence was determined to be at least 1533 bp long (Figure 1B) and was deposited in GenBank (accession number AF144103) formerly under the name NJAC. Continuity between ESTs with the ORF and the sequence of the differentially expressed gene fragment N0402A (shown as the underlined sequence in Figure 1B ) was confirmed by PCR amplification and sequencing of the contiguous cDNA fragment. The ORF predicted a protein 111 amino acids long, with two potential methionine translation start sites separated from each other by just 11 amino acids. Starting from the second methionine start site, the protein is 100% identical to the recently cloned BRAK (GB AF073957) gene, which encodes a precursor protein of 99 amino acids.¹ BRAK belongs to the CXC subfamily, all members of which contain a total of four conserved cysteine resides, with the first two separated by a nonconserved amino acid. At the nucleic acid level, the differentially expressed gene and BRAK were also 100% identical over the region encoding the second methionine residue to a stretch of "A" residues following the TAG termination codon; however, our sequence contained an additional 1066 untranslated nucleotides that were not determined for BRAK. The theoretical cleavage site of the BRAK signal peptide was predicted to occur immediately before the sequence SKCK, using the SignalP V1.1 software available at http://www.cbs.dtu.dk/services/SignalP/ (regardless of which upstream methionine start site was chosen) and is thus identical to the cleavage site previously predicted.¹

View larger version (55K): [in this window] [in a new window]   Figure 1. Molecular cloning of BRAK. A: Portion of a differential display gel comparing gene fragments from short-term explants of normal epithelial cells (N1) and squamous tumor cells (T1) derived from patient 1 with SCC of the tongue. B: Complete cDNA sequence of BRAK with predicted signal peptide (in parentheses) and amino acids of mature chain. The two potential methionine start sites appear in larger font. The asterisk indicates the stop codon, and the underlined sequence was derived from cloning and sequencing gene fragment N0402A. C: Schematic diagram of the genomic structure of the BRAK gene. The size interval between introns is indicated but is not to scale with the size of exons.

A search of the STS database using the entire BRAK cDNA yielded a 99% match between the human STS marker TIGR-A002I14 (which maps to chromosome 5q23–5q31) and a 120-bp stretch of BRAK cDNA from bp 1255 to bp 1314, which was previously unpublished. This is the same chromosomal area that Hromas et al¹ mapped BRAK to, based on sequence homology between nucleotides in the open reading frame and a genomic BAC clone. The STS marker TIGR-A002I14 localizes to the BAC clone AC005738 that contains 134 kb of genomic sequence from human chromosome 5 and encodes the entire BRAK gene. Alignment between BRAK cDNA and the genomic sequences in the BAC clone identified four exons that span an 8.1-kb region of genomic DNA (Figure 1C) . Exon and intron boundaries were confirmed using the HSPL program that predicts RNA splice sites (available on the web through the Baylor College of Medicine Search Launcher at http://kiwi.imgen.bcm.tmc.edu:8088/search-launcher/launcher.htm). Exon 1 is at least 100 bp, includes two potential "ATG" start sites, and encodes for all but one of the amino acids in the putative signal peptide. Exons 2 (106 bp) and 3 (115 bp) encode the majority of the remaining BRAK peptide, and exon 4, which is the largest (1182 bp), encodes the terminal four amino acids, stop codon, and potential polyadenylation site. A search of the genomic sequences 1650 bp upstream of the translational start site did not reveal any interferon response elements as are found in IP-10; however, several AP-2 and Sp1 binding sites were detected.

Phylogenetic Relationship of BRAK to Other CXC Chemokine Family Members

A multiple sequence alignment comparing the predicted mature BRAK peptide with the 13 other known human CXC chemokines was performed and used to generate the phylogenetic tree in Figure 2 . BRAK has the greatest homology with Mig (29% amino acid identity) and appears to have diverged from most CXC chemokines very early in evolution.

View larger version (25K): [in this window] [in a new window]   Figure 2. Phylogenetic relationship of all known human CXC chemokines. A ClustalW alignment of BRAK with known human CXC genes was used to generate the phylogenetic tree. The numbers in parentheses indicate the percentage amino acid identity with BRAK.

A fragment of BRAK cDNA corresponding to the entire coding region was used to probe a Northern blot containing total RNA extracted from cultured cells as well as flash-frozen biopsy specimens from HNSCC patients (Figure 3A) . A single 1.5-kb band corresponding to BRAK mRNA was evident in RNA extracted from both cultured normal oral epithelial cells and cultured human normal epidermal keratinocytes (HNEKs). BRAK mRNA was completely absent from a panel of seven established HNSCC tumor lines. To determine whether the differential expression might have been due to the different culture mediums used, two HNSCC lines (Tu-138 and Tu-177) were cultured for 30 hours with KGM medium (used for growing normal oral epithelial cells and keratinocytes). BRAK mRNA was undetectable from these tumor cell lines, regardless of their growth in KGM medium. As expected, BRAK mRNA was also detected in RNA extracted from a nonneoplastic normal tongue epithelial tissue biopsy. However, BRAK was found in specimens from two patients with SCC of the tongue (patient 1) and buccal region (patient 2). The results of the Northern blot in Figure 3A have been confirmed by relative RT-PCR in separate experiments (data not shown).

View larger version (59K): [in this window] [in a new window]   Figure 3. Detection of BRAK mRNA by Northern blotting. A: Northern analysis of BRAK expression in short-term explants of normal oral epithelial (NOE) cells, cultured normal epidermal keratinocytes (HNEKs), established HNSCC lines grown in regular or KGM growth medium, and biopsy specimens, including normal tongue epithelium (patient 1), invasive HNSCC of the tongue (patient 1), and invasive HNSCC of the buccal region (patient 2). B: Multiple tissue-Poly A mRNA Northern blot probed for BRAK (top) or stripped and reprobed with a sequence complementary to ß-actin. In some tissues, the ß-actin probe also cross-hybridizes to -actin.

Several CXC chemokine family members are up-regulated by IL-1ß, TNF, or IFN-. Therefore, the ability of these cytokines to up-regulate BRAK expression in HNEKs was examined in vitro. No increased expression of BRAK mRNA was apparent after incubation with any of these cytokines, regardless of whether HNEKs were cultivated in complete KGM medium or basal medium lacking the usual growth factor additives (data not shown).

In Situ Detection of BRAK mRNA in Head and Neck Tissue

Because HNSCC lines were all negative for BRAK message, in situ mRNA hybridization studies were undertaken to identify which cell populations within tumors and adjacent nonneoplastic tissues were expressing this chemokine. A digoxigenin-labeled antisense RNA probe to BRAK was hybridized to paraffin sections containing tumor or adjacent nonneoplastic tissue from HNSCC patients; a summary of the results appears in Table 1 . Strong expression of BRAK mRNA was detected in all layers of nonneoplastic squamous epithelium (basal to superficial) derived from the tongue and buccal mucosa and anatomically as far caudal as the true vocal cord of the larynx (Figure 4) . Although most of the nonneoplastic tissues were adjacent to cancerous areas and theoretically could have been altered, in at least one case the normal tongue biopsy specimen expressing BRAK (Figure 4A) was derived from a patient with a contralateral buccal SCC. In addition, murine BRAK is 98% identical to the human gene at the amino acid level,¹ and we have also been able to detect its expression in normal mouse tongue from undiseased animals by Northern blotting (data not shown). BRAK mRNA was absent from nine of 13 HNSCCs (Table 1) derived from areas of the tongue (Figure 4C) , buccal mucosa (Figure 4L) , pharynx, and larynx (Figure 4J) . Strong expression of BRAK mRNA in inflammatory cells (Figure 4D) adjacent to tumors was a frequent finding (ie, five of 15 cases) in HNSCC, and high levels of mRNA occasionally occurred in stromal fibroblasts (Figure 4K) adjacent to tumors as well. Expression was also observed in dysplastic epithelium from the tongue (Figure 4B) and occasionally in endothelial cells (data not shown). The in situ hybridization data were consistent with absence of expression in the panel of HNSCC cell lines and suggest that BRAK expression may be down-regulated or lost by tumor cells during development or progression of cancer in vivo.

View larger version (148K): [in this window] [in a new window]   Figure 4. In situ hybridization analysis of BRAK mRNA expression in head and neck tissue. A digoxigenin-labeled antisense (A–D and I–L) or control sense (E–H and M–P) probe generated from the middle portion of BRAK cDNA was hybridized to paraffin sections. A: Detectable BRAK expression in normal tongue. B: BRAK expression in a dysplastic region of the tongue. C: Absence of BRAK from a metastatic SCC of the tongue. D: Numerous inflammatory cells expressing BRAK in the vicinity of a SCC of the tongue (tumor not visible). E–H: Sense controls of corresponding regions in consecutive sections. I: Detection of BRAK in normal squamous epithelium from the true vocal cord. J: Absence of BRAK from a laryngeal SCC. K: Numerous inflammatory and stromal cells, showing expression of BRAK adjacent to a buccal SCC (bottom of picture) that does not make the chemokine. L: Absence of BRAK from a buccal SCC. M–P: Sense controls of the corresponding regions stained on consecutive sections. Original magnifications, x200.

Although BRAK was originally identified from ESTs derived from breast and kidney cancer libraries, in vitro expression was absent from a panel of established breast cancer cell lines.¹ Therefore, the cell types expressing BRAK mRNA were examined in cancerous and nonneoplastic tissues derived from breast and other gynecological sites. Table 2 summarizes the results of in situ mRNA hybridization in these tissues. Although the vast majority of normal breast lobules were negative, weak expression of BRAK could be detected in an isolated area of normal breast lobules (Figure 5A) . Within these lobules, expression was mostly confined to the inner luminal epithelium. No BRAK mRNA was detected in five of five invasive ductal carcinomas of the breast (eg, Figure 5B ). However, strong expression of BRAK mRNA was detected in inflammatory and stromal cells immediately adjacent to some of the breast carcinomas (Table 2) .

View larger version (150K): [in this window] [in a new window]   Figure 5. In situ hybridization analysis of BRAK mRNA expression in other tissues. A digoxigenin-labeled antisense (A–D and I–L) or control sense probe (E–H and M–P) generated from the middle portion of BRAK cDNA was hybridized to paraffin sections. A: Weak expression of BRAK in a cluster of normal breast lobules. B: Absence of BRAK in an invasive breast ductal carcinoma. C: Detection of BRAK in normal squamous epithelium from the exocervix of a healthy volunteer. D: Heterogeneous expression of BRAK in a cervical SCC. E–H: Sense controls of corresponding regions in consecutive sections. I: Detection of BRAK in normal transitional epithelium from the renal calyx. J: A transitional squamous carcinoma from the renal pelvis expressing BRAK. K: Absence of BRAK from normal columnar epithelium of the colon. L: Weak BRAK expression in a colon adenocarcinoma. M–P: Sense controls of the corresponding regions stained on consecutive sections. Original magnifications, x200.

In Situ Detection of BRAK mRNA in Kidney and Colorectal Tissues

On the multitissue blot in Figure 3B , high levels of BRAK were found in the kidney and colon. It was therefore of interest to determine the cell types responsible for BRAK expression in these tissues. Although BRAK was also highly expressed in the small intestine, this region was excluded from our in situ analysis, as cancers from this site are uncommon. Table 3 summarizes the findings in kidney and colorectal tissue. Expression in normal proximal and distal tubules of the kidney was consistently detected but was sometimes weak. BRAK mRNA was strongly detected in nonneoplastic transitional epithelium of the renal calyx (Figure 5I) that was adjacent to a transitional cell carcinoma of the renal pelvis, which also expressed high levels of BRAK (Figure 5J) . Renal cell carcinoma of the clear type is by far the most frequent kidney tumor that occurs; however, these tumors were all negative for BRAK mRNA by in situ hybridization (Table 3) . As with the other cancers, BRAK mRNA was found in inflammatory and stromal cells adjacent to the renal carcinomas.

Morphology of Inflammatory Cell Populations Expressing BRAK

Expression of BRAK in inflammatory cells adjacent to tumors was a frequent finding. Examples of inflammatory cells expressing BRAK from cancer of the soft palate, colon, and breast are shown in Figure 6 . Hematoxylin and eosin (H&E) staining of consecutive sections from these regions revealed numerous inflammatory cells with lymphoid morphology. In the soft palate specimen there were numerous plasma cells and small lymphocytes (Figure 6J) . However, the majority of inflammatory cells present in the colon adenocarcinomas had a morphology consistent with small lymphocytes (eg, Figure 6K ). In the breast cancers as well, there were predominantly small lymphocytes in the inflammatory areas expressing BRAK (eg, Figure 6L ).

View larger version (107K): [in this window] [in a new window]   Figure 6. BRAK expression by infiltrating inflammatory cells in various tissues. A digoxigenin-labeled antisense (A–C) or control sense (D–F) probe generated from the middle portion of BRAK cDNA was hybridized to paraffin sections. The same regions were stained with hematoxylin and eosin (H&E) on consecutive sections. Original magnifications: G–I, x200; J–L, x400. A: Numerous inflammatory cells expressing BRAK adjacent to a soft palate SCC (tumor not visible). B: Detection of BRAK in scattered inflammatory cells adjacent to a colon adenocarcinoma (tumor not visible). C: Expression of BRAK by inflammatory cells that have infiltrated a breast ductal carcinoma that is itself negative for BRAK. G–I: H&E stains of corresponding regions. J: At higher magnification, many small and larger lymphocytes can be seen to be localizing to the same area where inflammatory cells positive for BRAK were evident adjacent to the soft palate SCC. An arrowhead indicates a small lymphocyte, and the full arrow points to an example of a plasma cell. K: Higher magnification of inflammatory cells adjacent to the colon adenocarcinoma. An example of a cell with morphology indicative of a small lymphocyte is shown by the arrowhead. L: Higher magnification of inflammatory cells infiltrating the breast ductal carcinoma. An example of a cell with morphology indicative of a small lymphocyte is shown by the arrowhead.

Because in situ mRNA hybridization consistently demonstrated expression of BRAK in inflammatory cells with the morphology of lymphocytes, we sought further proof that this subpopulation of cells was capable of making BRAK mRNA. The riboprobe used to detect BRAK could not be used successfully in combination with immunohistochemistry techniques. Therefore, the question was addressed in vitro. Freshly isolated PBMCs from healthy volunteers were cultured with various stimuli, and extracted RNA was examined for the presence of BRAK mRNA by RT-PCR, using BRAK-specific primers from exons II and IV. A 236-bp BRAK-specific fragment was amplified by RT-PCR from PBMCs cultured in the presence of either PMA/calcium-ionophore or LPS, but not other stimuli (Figure 7A) . No BRAK expression was detected in RNA extracted from freshly isolated PBMCs or after overnight culture in medium containing 10% FBS. The plasmid pCMV-BRAKfg, which contains the entire coding portion of cDNA from the BRAK gene, served as a gene-specific positive control. The integrity of the cDNA used in PCR reactions was confirmed by amplifying a portion of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Figure 7A , bottom gel). As there were a number of biopsy specimens in which plasma cells (B cells) were detected by H&E staining in the vicinity of BRAK-positive inflammatory cells (eg, Figure 6J ), it was possible that B cells were making BRAK mRNA after in vitro stimulation of PBMC with LPS. Consequently, PBMCs were stimulated with LPS for 6 hours and then separated into T-cell, monocyte, and B-cell subpopulations, using a flow cytometer based on expression of the cell surface markers CD3 (T cells), CD33 (monocytes), and CD19 (B cells). The monocyte marker CD33 was chosen in lieu of CD14 to sort monocytes, because the latter recognizes the LPS receptor and could have confounded interpretation of the results. Preliminary analysis indicated that the CD33 marker was present on 85% of CD14+ monocytes before and after stimulation with LPS and was not expressed by T cells or B cells (data not shown). Granulocytes that can also express CD33 were removed from the starting population during purification of PBMC on Histopaque gradients, and any residual granulocytes were gated out during sorting. Because of limiting amounts of RNA, RT-PCR with BRAK-specific primers was again used to detect BRAK expression. After stimulation with LPS, BRAK RNA was detected in unsorted PBMC, B cells, and monocytes, but not in the T-cell population (Figure 7B , top gel). GAPDH, however, was detected in all populations examined (Figure 7B , bottom gel).

View larger version (32K): [in this window] [in a new window]   Figure 7. RT-PCR detection of BRAK in stimulated PBMCs. A: PBMCs (2 x 106/ml) were stimulated for 8 hours with various agents as described in Materials and Methods, and extracted RNA was analyzed by RT-PCR for the presence of a BRAK message, using gene specific primers (top gel). Plasmid pCMV-BRAKfg contains the entire coding region of BRAK cDNA and was used as a positive control. To control for the integrity of cDNA synthesis and PCR amplification, the same RT reaction was also subjected to PCR to amplify GAPDH (bottom gel). B: PBMCs (2 x 106/ml) were stimulated for 6 hours with 0.5 µg/ml LPS and then sorted into populations of T cells, B cells, or monocytes, based on the expression of cell surface markers. RNA was extracted from unsorted or purified populations and subjected to RT-PCR analysis as in A. The negative control (Neg. Crl) reaction was amplified without cDNA to rule out cross-contamination.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Note Added in Proof References

In the normal tissues examined, the predominant cell type constitutively expressing the highest levels of BRAK mRNA was squamous cell epithelium from the upper aerodigestive tract and exocervix. We have also detected high levels of BRAK mRNA in normal squamous epithelium from the skin (data not shown). Expression was found to extend from the basal to the superficial layers of squamous epithelium in both mucosal and skin tissues. In contrast, columnar epithelium from the endocervix and colon failed to express BRAK. There was weak and focal expression in normal epithelium that lines breast lobules. Distal and proximal tubules of the kidney consistently expressed BRAK, but the levels were sometimes weak. The latter results may explain the relatively strong signal for BRAK mRNA that we and Hromas et al¹ detected in kidney tissue on poly A⁺ mRNA Northern blots. As tubules are a main component of the kidney, even modest amounts of BRAK mRNA at the cellular level could produce a strong signal in whole tissue homogenates. However, the ubiquitous expression of BRAK mRNA in other tissues on poly A⁺ blots could be due to contaminating cell populations present in whole tissue extracts. It is clear that BRAK mRNA can be made at high levels by inflammatory and stromal cells.

Normal human lymphocytes from blood do not express BRAK mRNA by Northern analysis or even by the more sensitive technique of RT-PCR. Yet high levels of BRAK were consistently detected in infiltrating inflammatory cells with lymphocyte morphology in the vast majority of cancers we studied. This would suggest that factors in the environment of the tumor could be up-regulating BRAK expression. In freshly isolated PBMCs from healthy volunteers, BRAK expression was detectable by RT-PCR after stimulation with either LPS or PMA/calcium ionophore. When LPS-stimulated PBMCs were separated by flow cytometry, BRAK expression was found in both B-cell and monocyte populations, but not in CD3+ T cells. These in vitro experiments provide independent corroborative evidence that inflammatory cells are capable of BRAK expression. Furthermore, plasma cells (B cells) were frequently found in consecutive sections in the immediate vicinity of inflammatory cells positive for BRAK by in situ mRNA hybridization and therefore could be one of the leukocyte subtypes expressing BRAK mRNA in cancerous tissues. Final confirmation of the inflammatory cell phenotype expressing BRAK will have to await the production of a BRAK-specific antiserum that can be used in immunohistochemistry. It is currently unknown which population of cells expresses BRAK after stimulation of PBMCs with PMA/calcium ionophore, and it cannot be ruled out that T cells or natural killer cells could express BRAK given the appropriate stimulus. The ability of local factors to up-regulate BRAK may also explain why certain colon adenocarcinomas were capable of expressing the chemokine, despite the fact that adjacent normal columnar epithelium (which presumably gives rise to these cancers) did not express BRAK.

In vivo expression of BRAK mRNA in tumors was more heterogeneous than would be predicted from our own data with established tumor cell lines and the study from Hromas et al.¹ Because squamous epithelium appears to be a predominant cell type constitutively expressing BRAK mRNA, issues regarding whether down-regulation occurs in cancer are best studied in tumors derived from these cells. Most head and neck cancers originate from squamous epithelium, and the majority of cervical cancers are SCCs that arise from the squamocolumnar junction of the exocervix. Normal squamous epithelium from both of these regions constitutively expressed BRAK. Yet the majority of HNSCCs and two of six cervical SCCs failed to express BRAK mRNA. Thus there does indeed appear to be loss of BRAK mRNA occurring in certain tumors. Most breast cancers, however, arise from the same epithelium that gives rise to normal lobular units. As the in vivo expression in normal lobular units was rare, it would not necessarily be expected for breast adenocarcinomas to produce BRAK mRNA. This is also true of ovarian cancer, because the normal ovary does not appear to express BRAK either. On the other hand, expression in colon cancer had an interesting pattern. The normal columnar epithelium (that gives rise to the majority of adenocarcinomas) had no BRAK expression, yet in some cases there was weak or even strong expression in the corresponding colorectal tumors. That coexpression of BRAK mRNA in subpopulations of inflammatory cells was found in 100% of the colorectal cancers studied raises the possibility that BRAK mRNA could even be up-regulated in certain tumors because of locally produced factors.

At present, the role of BRAK in the biology of tumors is unknown and there are no existing data regarding the function of an active recombinant protein. However, three lines of evidence suggest that there could be some involvement in host-tumor interactions. First, down-regulation of BRAK was observed in certain tumors. Second, up-regulation of BRAK was a ubiquitous finding in infiltrating inflammatory cells in numerous cases. Third, there is increasing evidence implicating chemokines in the regulation of tumor growth.^{2;8-12 10-13} The chemokines MCP-1,¹⁴ mTCA3,¹⁵ RANTES,¹⁶ IP-10,¹³ and Mig¹⁷ have all been shown to cause tumor regression or slow growth of tumors in mice when transfected into various tumor cell lines or directly administered to mice. In contrast, expression of GRO, and IL-8 leads to increased tumorigenicity.^18;19

BRAK belongs to the CXC chemokine subfamily, certain members of which have been shown to regulate the angiogenesis needed for solid tumor growth.^2;20-22 CXC chemokines can be subdivided according to the presence or absence of the amino acid motif "glutamic acid-leucine-arginine (ELR)". Several ELR⁺ CXC chemokines stimulate migration of endothelial cells in vitro and induce angiogenesis in vivo, whereas ELR^- chemokines inhibit endothelial cell chemotaxis and block angiogenesis mediated by ELR⁺ chemokines, basic fibroblast growth factor, and vascular endothelial growth factor.^20-22 Evidence that the ELR motif determines whether CXC chemokines inhibit or stimulate angiogenesis comes from studies employing recombinant chimeric proteins.²¹ BRAK lacks the ELR motif and may therefore turn out to be an angiogenesis inhibitor.

A plethora of data exists supporting the idea that the balance of angiogenic and angiostatic chemokines can alter the in vivo growth of tumors.^12;18;20;22 Recently, the antitumor properties of IL-12 have been shown to be mediated by secondary induction of the angiostatic CXC chemokines IP-10 and Mig in both immunocompetent¹¹ and T-cell-deficient mice.¹² While the angiostatic behavior of certain CXC chemokines is a plausible explanation for their antitumor properties, there also appears to be an immunological mechanism in some cases. Murine plasmacytomas and mammary carcinomas transfected with the CXC chemokine IP-10 become infiltrated by lymphocytes and are rejected by immunocompetent mice but not by immunodeficient nude mice.¹⁰ Further studies are needed to determine whether BRAK behaves like an angiogenesis inhibitor or simply like a chemotactic factor. The fact that many other CXC family members regulate tumor growth is intriguing, because BRAK expression is lost in certain tumors and is frequently expressed by infiltrating inflammatory cells. An understanding of the biological significance, however, must await definition of the functional properties belonging to this novel chemokine.

	Note Added in Proof

Top Abstract Introduction Materials and Methods Results Discussion Note Added in Proof References

 
While this manuscript was in press, the murine homologue of BRAK (BMAC) was described and found to stimulate chemotaxis of B cells and monocytes. Sleeman MA, Fraser JK, Murison JG, Kelly SL, Pestidge RL, Palmer DJ, Watson JD, Kumble KD: B cell- and monocyte-activating chemokine (BMAC), a novel non-ELF -chemokine. Int Immunol 2000, 12:677–689.

	Acknowledgements

 
We thank Drs. Michael Hudson, Michael Gilcrease, Marc Levy, and Jeffrey Scott for their competent technical assistance, and Paula Holton for organizing and coordinating the procurement of specimens.

	Footnotes

 
Address reprint requests to Dr. Gary L. Clayman, Department of Head and Neck Surgery, Box 69, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe, Houston, TX 77030.

Supported in part by grants from the National Institute of Dental Research 1-P50-DE11906 (93-9), a National Institute of Health First Investigator Award (R29 DE11689-01A1), a Training of the Academic Head and Neck Surgical Oncologist Core support grant (T32 CA60374–03 to G. L. C.), the Betty Berry Cancer Research Fund, the Michael A. O’Bannon Foundation Cancer Research Fund, a Cancer Center grant (NIH-NCI-CA16672), and a Physicians Referral Service grant from The University of Texas M. D. Anderson Cancer Center.

Accepted for publication February 16, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion Note Added in Proof References

 

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