Presence of a new conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is expressed in breast carcinoma.

CART1, a novel human gene, encodes a putative protein exhibiting three main structural domains: first, a cysteine-rich domain located at the amino-terminal part of the protein, which corresponds to an unusual RING finger motif; second, an original cysteine-rich domain located at the core of the protein and constituted by three repeats of an HC3HC3 consensus motif that we designated the CART motif, and which might interact with nucleic acid; third, the carboxyl-terminal part of the CART1 protein corresponds to a TRAF domain known to be involved in protein-protein interactions. Similar association of RING, CART, and TRAF domains was observed in the human CD40-binding protein and in the mouse tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), both involved in signal transduction mediated by the TNF receptor family and in the developmentally regulated Dictyostelium discoideum DG17 protein. CART1 is specifically expressed by epithelial cells in breast carcinomas and metastases. Moreover, in these malignant cells, the CART1 protein is localized in the nucleus. Altogether, these observations indicate that CART1 may be involved in TNF-related cytokine signal transduction in breast carcinoma.

Despite earlier detection and a lower size of the primary tumors at the time of diagnosis (Nystrom et al., 1993;Fletcher et al., 1994), breast cancer mortality remains high (Frost and Levin, 1992). Therefore, defining the molecular mechanisms involved in cancer formation and progression is still a major challenge in breast cancer research (Rusciano and Burger, 1992;Hoskins and Weber, 1994). Human CART1 cDNA corresponds to the metastatic lymph node 62-cDNA clone recently isolated from a cDNA library of breast cancer-derived metastatic lymph nodes. 10 5 recombinants were differentially screened using two subtractive probes, respectively, represent-ative of malignant (metastatic lymph node) and non-malignant (fibroadenoma) breast tissues, to identify new genes that might be specifically involved in breast cancer (Tomasetto et al., 1995). 2% of the clones differentially expressed contained CART1 cDNA insert. CART1 has been mapped on the q11-q12 region of the long arm of chromosome 17 (Tomasetto et al., 1995), a locus that includes the oncogene c-erbB2 whose overexpression is correlated with a shorter overall and disease-free survival for breast cancer patients (Slamon et al., 1987;Muss et al., 1994).
In the present study, we characterized the CART1 cDNA, protein, and gene organization and investigated CART1 gene expression in a panel of normal and malignant human tissues. CART1 was specifically expressed in epithelial breast cancer cells. The predicted amino acid sequence of CART1 reveals a new conserved cysteine-rich domain that we name the CART domain. Moreover, the CART1 protein showed a structural organization similar to that present in the recently identified TNF receptor-associated proteins (Rothe et al., 1994;Hu et al., 1994). Finally, in breast carcinoma, CART1 protein is localized in the nucleus of the malignant cells. Altogether, these results suggest that CART1 is implicated in signal transduction by TNF-related cytokines maybe as a latent cytoplasmic transcription factor.

MATERIALS AND METHODS
Tissue Collection-Depending on subsequent analysis, tissues were either immediately frozen in liquid nitrogen (RNA extraction) or fixed in formaldehyde and paraffin embedded (in situ hybridization). Frozen tissues were stored at Ϫ80°C, whereas paraffin-embedded tissues were stored at 4°C.
The mean age of the 39 patients included in the present study was 55 years. The main characteristics of the breast carcinomas were as follows: SBR grade I (13%), grade II (38%), and grade III (49%); estradiol receptor positive (25%) and negative (75%); and lymph nodes without invasion (39%) and with invasion (61%).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) X80200.
Preparation of Polyclonal Antibodies and Immunohistochemistry-The QSDPGLAKPQHVTETFHPD (residues 393-411) synthetic peptide, corresponding to a CART1-specific sequence of the TRAF-C domain (Figs. 1 and 5), was synthesized in solid phase using Fmoc (N-(9fluorenyl)methoxycarbonyl) chemistry (model 431A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA), verified by amino acid analysis (model 420A-920A-130A analyzer system, Applied Biosystems, Inc.), and coupled to ovalbumin (Sigma) through an additional NH 2extraterminal cysteine residue, using the bifunctional reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester (Aldrich). Two rabbits were injected subcutaneously with 200 g of coupled antigen every 2 weeks until obtention of positive antisera. Immunohistochemical analysis was performed on paraffin-embedded tissue sections using a peroxidaseantiperoxidase system (DAKO, Carpinteria, CA) for the revelation as described previously (Rio et al., 1987).
Sequencing Reactions-CART1 cDNA clones and genomic subclones prepared as described (Zhou et al., 1990) were further purified with RNase A treatment (10 g/ml; 30 min, 37°C) followed by polyethylene glycol/NaCl precipitation (0.57 volume; 20%, 2 M) and ethanol washing. Vacuum-dried pellets were resuspended at 200 ng/l in TE. Doublestranded DNA templates were then sequenced with Taq polymerase, using pBluescript universal primers and/or internal primers, and dye labeled dNTPs for detection on an Applied Biosystems 373A automated sequencer.
Computer Analysis-Sequence analyses were performed using the GCG sequence analysis package (Wisconsin Package, version 8, Genetic Computer Group). The CART1 cDNA sequence and its deduced putative protein were used to search the complete combined GenBank/EMBL data bases and the complete SwissProt data base, respectively, with BLAST (Altschul et al., 1990) and FastA (Pearson and Lipman, 1988) programs. The RING finger motif and consensus sequences of CART1 protein were further identified by the Motifs program in the PROSITE dictionary (release 12). The sequence alignments were obtained automatically by using the program PileUp (Feng and Doolittle, 1987).
CART1 Genomic DNA Cloning-50 g of human genomic DNA was Sau3A partially digested. After size selection on a 10 -30% sucrose gradient, inserts (16 -20 kb 1 ) were subcloned at the BamHI replacement site in EMBL 301 (Lathe et al., 1987). 2.5 10 6 recombinant clones were obtained, and the library was amplified once. 1 million plaque-forming units were analyzed for the presence of genomic CART1 DNA, using the full-length CART1 cDNA probe. 30 clones gave a positive signal. After a second screening, 4 of these clones were subcloned into pBluescript II SKϪ vector (Stratagene), sequenced, and positioned with respect to the CART1 cDNA sequence.

Determination of Human CART1 cDNA and Putative Protein
Sequences-The complete CART1 cDNA sequence has been established from three independent cDNA clones. Both sense and antisense strands have been sequenced. The longest cDNA clone contained 2004 base pairs, a size consistent with the previously observed 2-kb transcript, suggesting that this cDNA corresponded to a full-length CART1 cDNA (Fig. 1). The first ATG codon (at nucleotide position 85) had the most favorable context for initiation of translation (Kozak, 1987), and a classical AATAAA poly(A) addition signal sequence (Wahle and Keller, 1992) was located 18 base pairs upstream of the poly(A) stretch. Thus, the open reading frame was predicted to encode a 470-residue protein, with a molecular mass of 53 kDa and a calculated isoelectric point of 8. The putative protein showed several consensus sequences and notably two potential nuclear localization signals (NLS), a monopartite KPKRR (residues FIG. 1. cDNA and deduced amino acid sequences of human CART1. Nucleotide residues are numbered in the 5Ј to 3Ј direction, and amino acids in the open reading frame are designated by the one-letter code. The underlined nucleotide sequences correspond to the Kozak and poly(A) addition signal sequences. Putative NLS sequences are shown in bold type and broken underline. The two C-rich regions are boxed, and His and Cys residues are in bold type. TRAF-C domain is shown with a gray box. Arrow heads indicate the splicing sites, and the asterisk indicates the stop codon. 11-15) (Dang and Lee, 1989), and a bipartite RR-X 11 -KRRLK (residues 123-140) (Dingwall and Laskey, 1991). The molecule also contained potential sites (reviewed in Kemp and Pearson (1990)) specific of N-glycosylation (NGS, residues 355-357), phosphorylation by casein kinase I (EELS, residues 300 -303; SVGS, residues 303-306; ECFS, residues 331-334) and casein kinase II (SEE, residues 86 -88; SRRD, residues 122-125; SGE, residues 149 -151; SHE, residues 155-157; TSE, residues 185-187; TKE, residues 199 -201; SGE, residues 357-359; SLLD, residues 389 -392; SLDE, residues 426 -429; SHQD, residues 441-444), and proline-dependent phosphorylation (FSPA, residues 333-336) and cAMP-dependent phosphorylation (RRVT, residues 384 -387). Moreover, two cysteine-rich (C-rich) regions were identified, one located at the amino-terminal part of the protein (residues 18 -57) and the other at the core of the molecule (residues 83-282). Finally, the carboxylterminal part of the CART1 protein corresponded to the recently described TRAF domain (Rothe et al., 1994) (Fig. 1).
CART1 Contains an Unusual Amino-terminal RING Finger Motif-The amino-terminal C-rich structure of the putative CART1 protein contained a CX 2 CX 12 CX 1 HX 2 CX 2 CX 11 CX 2 D (C3HC3D) motif (residues 18 -57, Fig. 1) reminiscent of the C3HC4 consensus sequence (Freemont et al. (1991) and Fig. 2). This sequence, located either at the amino-or at the COOHterminal part of proteins has been named the RING finger motif (Freemont (1993) and references therein) and gives rise to two zinc fingers (Borden et al., 1995). The proteins that share such a structure have been reported to be implicated during development such as DG17 (Driscoll and Williams, 1987) and SU(z)2 (Van Lohuizen et al., 1991), gene transcription such as RPT-1 (Patarca et al., 1988), SS-A/Ro (Chan et al., 1991), XNF7 (Reddy and Etkin, 1991), and RING1 (Lovering et al., 1993), DNA repair such as RAD-18 (Jones et al., 1988), cell transformation such as MEL-18 (Tagawa et al., 1990;Goebl, 1991), tumor suppression such as BRCA1 (Miki et al., 1994), or signal transduction such as CD40-binding protein (CD40-bp) (Hu et al., 1994) and TRAF2 (Rothe et al., 1994). The distribution of Cys and His residues is highly conserved in all of these RING fingers (Fig. 2). However, CART1 contained an aspartic acid residue instead of the last Cys residue of the C3HC4 motif (Fig.  2). To confirm the presence of this Asp residue, and since Asp codon sequence leads to an AvaII restriction site (Fig. 3A), an AvaII digestion was performed on the full-length CART1 cDNA. Gel electrophoresis showed the presence of four bands (253, 428, 531, and 792 base pairs, respectively), a pattern consistent with the presence of an Asp codon (Fig. 3B). However, since the CART1 cDNA was cloned from a cDNA library established using malignant tissues (Tomasetto et al., 1995), we could not exclude the possibility that the Asp residue resulted from an alteration occurring during carcinogenesis (Bishop, 1991). Thus, to identify the physiological residue, we sequenced CART1 DNA from a normal leukocyte genomic library (see "Materials and Methods"). This analysis confirmed the presence of an Asp residue and consequently the C3HC3D motif. Data bank library analysis did not reveal any other protein sharing an identical RING finger motif.
Identification and Characterization of a Novel C-rich Domain, the CART Domain-The second C-rich region expanded from residue 83 to residue 282 and constituted almost half of the protein (Fig. 1). It contained 23 Cys and 12 His residues, corresponding to 96 and 67% of the remaining Cys and His residues, respectively. A careful examination of spacing of these Cys/His residues allowed the detection of an ordinance giving rise to three HX 3 CX 6 CX 3 CX 11-12 HX 4 CX 6 CX 2-6 CX 11 (HC3HC3) repeats. The most amino-terminal repeat (residues 101-154) contained the potential bipartite NLS (Figs. 1 and 4). Homologies between these repeats were not restricted to the Cys/His residues and to the spacer sizes. Alignment of the three CART1 HC3HC3 motifs showed around 50% similarity and 30% identity with each other (Fig. 4). Homology searches in the protein data base (see "Materials and Methods") revealed the presence of one copy of an analogous motif (residues 193-250) in the Dictyostelium discoideum DG 17 protein (Driscoll and Williams, 1987) and of two copies in the human CD40-bp (residues 134 -189 and 190 -248) (Hu et al., 1994) and in the mouse TRAF2 (residues 124 -176 and 177-238) (Rothe et al., 1994). It should be noted that the sequences of the two aminoterminal CART1 HC3HC3 motifs were most similar to those of the amino-terminal motifs of CD40-bp (50 and 40%, respectively) and of TRAF2 (52 and 46%, respectively). The COOHterminal CART1 HC3HC3 motif, however, was most similar to the COOH-terminal motifs of CD40-bp (58%) and of TRAF2 (55%) and to that of DG17 (51%) (Fig. 4). From these comparisons, the HX 3-4 CX 6 CX 2-4 CX 11-12 HX 3-4 CX 6 CX 2/6 CX 11 consensus sequence was proposed for this novel domain, which we named the CART domain for "C-rich motif associated with RING and TRAF (see below) domains" (Fig. 4).
CART1 Contains a COOH-terminal TRAF Domain-The TRAF domain, recently identified in the TNF receptor-associated factors 1 (TRAF1) and 2 (TRAF2), is involved in TNF signal transduction pathway. TRAF domains encompass the 230 COOH-terminal residues of these proteins and share 53% identity (Rothe et al., 1994). The TRAF motif was also reported in the CD40-bp, which associates with the cytoplasmic tail of CD40, another member of the TNF receptor family (Hu et al., 1994). The COOH-terminal part of CART1 (residues 267-470) showed two degrees of homology corresponding to the TRAF-N and the TRAF-C domains (Cheng et al., 1995). From structural predictions, the CART1 TRAF-N domain (residues 267-307) is supposed to give rise to an ␣ helix (Chou and Fasman, 1978). Such a structure, already proposed for the corresponding regions of TRAF1, TRAF2, and CD40-bp, is supposed to be involved in protein-protein interactions (Rothe et al., 1994;Hu et al., 1994). The TRAF-C domain of CART1 (residues 308 -470) showed a high degree of similarity and identity with the corresponding part of TRAF1 (60 and 42%), TRAF2 (69 and 47%), and CD40-bp (62 and 43%) (Fig. 5). Finally, since DG17 already contained an amino-terminal RING finger and a CART motif, we looked for the presence of a TRAF-C domain in its COOHterminal part. We observed 55% similarity and 30% identity between the last 150 residues of CART1 and DG17 (data not shown), suggesting that DG17 contains a primitive TRAF domain.
CART1 Gene Organization-Two independent clones have been selected from a screening of a human leukocyte genomic library using the full-length CART1 cDNA probe. These clones contained 3-and 3.2-kb BamHI fragments, which have been subcloned and partially sequenced to map splicing sites. The human CART1 gene was found to be split into 7 exons ( Fig. 6 and Table I). Comparison of the intron/exon boundaries showed that each corresponded to a canonical splice consensus sequence (Breathnach and Chambon, 1981). The total length of the CART1 gene is approximately 5.5 kb (Fig. 6). Analysis of the genomic structure of the RING finger domain revealed that it is encoded by two exons separated by the presence of an intronic sequence located between nucleotides 226 and 227 (Fig. 1). Thus, the C3HC2 and the Cys-Asp parts of the C3HC3D motif are encoded by exons 1 and 2, respectively (Fig.  6). The three CART motifs were encoded by three separate exons of 161 (exon 4), 161 (exon 5), and 156 (exon 6) base pairs, respectively ( Fig. 6 and Table I). In addition to their similar size, the three exons exhibited about 40% identity with each other, suggesting they have arisen by duplication of an ancestral exon (Dorit et al., 1990). Finally, the TRAF-N and TRAF-C domains were encoded by exon 7, which also encoded the 3Јuntranslated region.
Expression of the CART1 Gene-Using Northern blot analysis, we have studied CART1 gene expression in benign (16 fibroadenomas) and malignant (39 carcinomas and 5 metastatic axillary lymph nodes) human breast tissues. Hybridization with a CART1 cDNA probe gave a positive signal corresponding to CART1 transcripts with an apparent molecular size of 2 kb in four carcinomas and two metastases (Fig. 7, lanes  7, 11, 13, and 17 and data not shown). The fibroadenomas did not show CART1 expression above the basal level (Fig. 7, lanes  1-6). No CART1 transcripts were observed in normal human breast, axillary lymph node, heart, brain, skin, lung, stomach, colon, liver, kidney, and placenta (data not shown).
In situ hybridization, using an antisense CART1 RNA probe, was performed on primary breast carcinomas and axillary lymph node metastases. CART1 was expressed in the malignant epithelial cells, in in situ (Fig. 8C) and invasive (Fig. 8B) carcinomas, whereas tumoral stromal cells were negative. CART1 transcripts were homogeneously distributed among the positive areas (Fig. 8, B and C). Normal epithelial cells did not express the CART1 gene, even when located at the proximity of invasive carcinomatous areas ( Fig. 8A and data not shown). A similar pattern of CART1 gene expression was observed in metastatic axillary lymph nodes from breast cancer patients with expression limited to cancer cells, whereas non-involved lymph node areas were negative ( Fig. 8D and data not shown).
CART1 Protein Subcellular Localization-CART1 subcellular localization was performed on paraffin-embedded sections from a human invasive breast carcinoma (Fig. 9A) using a rabbit polyclonal antibody. The antibody specificity was established by Western blot analysis of CART1 recombinant protein (data not shown). Consistent with our findings using in situ hybridization, CART1 immunoperoxidase staining (brown staining) was observed in malignant epithelial cells. Moreover, CART1 protein appeared to be located in the nucleus (Fig. 9B), showing that almost one of the CART1 nuclear localization signals was functional. The intensity of staining was variable from one cell to another, even within a given area of the section.

DISCUSSION
In the present study, we have characterized the products of a novel human gene that we called the CART1 gene, since it encodes a protein containing a new conserved C-rich domain associated with RING and TRAF domains (the CART domain).
The CART1 amino-terminal part contained a C-rich domain characterized by the presence of a RING finger motif giving rise to two zinc fingers (Freemont, 1993). The RING finger protein family presently comprises more than 70 members involved in the regulation of cell proliferation and differentiation (reviewed in Freemont (1993)). Interestingly, one of the recently identified members of the family is the tumor suppressor gene BRCA1, responsible for about 50% of inherited breast cancers FIG. 4. Primary structure of the three original HC3HC3 CART motifs present in CART1 and comparison with those of CD40-bp, TRAF2, and DG17. Alignment and conventional symbols are as described in the legend to Fig. 2. (Miki et al., 1994). In the CART1 RING finger, the last Cys residue is substituted by an Asp residue giving rise to a C3HC3D motif instead of the usual C3HC4 motif. Since aspartic acid has already been described as a potential zinc-coordinating residue (Vallee and Auld, 1990), we assume that the C3HC3D motif may efficiently bind metal atoms through the zinc-finger structure (Borden et al., 1995). Consistent with this hypothesis, aspartic acid has already been reported to be functional in another type of zinc-finger motif, the LIM domain (Sà nchez-Garcia and Rabbits (1994) and references therein). This represents the second variant of the RING finger domain since a C2THC4 motif has already been reported in the RING finger of the p53-associated oncoprotein MDM2 (Boddy et al., 1994). The CART1 RING finger is encoded by two exons coding for the C3HC2 and Cys-Asp part of the C3HC3D motif, respectively, a genomic organization slightly different from that previously described for the consensus MEL-18 RING finger, which results from two exons encoding the C3H and C4 putative zinc finger, respectively (Asano et al., 1993).
CART1 also contained an original C-rich region, located more centrally within the protein and composed of three repeats of an HC3HC3 motif corresponding to a novel protein signature, which we designated the CART motif. These three repeats were encoded by distinct exons homologous with each other, suggesting that they derived from an ancestral exon (Dorit et al., 1990). CART motifs were only found, in variable copy numbers, in three RING finger proteins, the human CD40-bp (two copies), the mouse TRAF2 (two copies), and the D. discoideum DG17 protein (one copy) (Hu et al., 1994;Rothe et al., 1994;Driscoll and Williams, 1987). The corresponding C-rich regions of CD40-bp (Hu et al., 1994;Cheng et al., 1995), TRAF2 (Rothe et al., 1994), and DG17 (Driscoll and Williams, 1987) have been previously reported to be partially arranged in a pattern resembling either the CHC3H2 "B box" motif or the C2H2 Xenopus laevis transcription factor III A motif. The CART motif, as defined in the present study, encompasses almost the totality of the corresponding C-rich region observed in CART1, CD40-bp, TRAF2, and DG17. The function of the CART domain remains to be determined. Preliminary protein studies 2 indicate that the correct folding of the CART motif is depending on the presence of zinc, supporting the hypothesis that CART corresponds to a novel zinc binding motif presumably involved in nucleic acid binding (Schwabe and Klug, 1994;Schmiedeskamp and Klevit, 1994).
The COOH-terminal part of CART1 corresponded to a TRAF domain previously identified in TRAF1, TRAF2, and CD40-bp. This motif is involved in protein-protein interaction; TRAF1, TRAF2, and CD40-bp have been reported to specifically interact with the cytoplasmic domain of two members of the TNF receptor family, TNF-R2 and CD40 (Rothe et al., 1994;Hu et al., 1994). The TRAF domain is composed of two structural domains, TRAF-N, which is amino-terminally located and corresponds to a weakly conserved ␣ helix, and TRAF-C, which is COOH-terminally located and highly conserved (Cheng et al., 1995). Both structural domains were encoded by the same exon 2 C. H. Régnier, unpublished data.   lanes 1-6), carcinomas (BC,, and metastatic lymph nodes (MLN, lanes 17 and 18) are loaded. Hybridization was carried out using 32 P-cDNA probe for CART1. A 2000-base-long CART1 transcript is expressed, at various levels, in some carcinomas (lanes 7, 11, and 13) and in one metastatic sample (lane 17). The 36B4 probe (Masiakowski et al., 1982) was used as a positive internal control. Autoradiography was for 2 days for hybridization of CART1, whereas 36B4 hybridization was exposed for 16 h. of the CART1 gene. Homology was also observed with the COOH-terminal part of the protozoan DG17 protein which, although less conserved, could be considered as a TRAF-C domain.
Thus, CART1 shared a protein organization similar to that of the human CD40-bp, the mouse TRAF 2, and the protozoan DG17, including an amino-terminal RING finger, one to three central CART motifs, and a COOH-terminal TRAF domain (Fig. 10). These results suggest that these structurally related proteins belong to the same protein family and may exhibit analogous function. DG17 is expressed during D. discoideum aggregation, which occurs under stress conditions to permit cell survival through a differentiated multicellular organism. The precise function of DG17 remains unknown (Driscoll and Williams, 1987). However, both CD40-bp and TRAF2 have been previously shown to be involved in TNF-related cytokine signal transduction (Hu et al., 1994;Rothe et al., 1994). In contrast to growth factor receptors, cytokine receptors generally do not contain kinase activity in their cytoplasmic region, and their signal transduction mechanisms remain elusive (reviewed by Taga and Kishimoto, 1993). To date, the TNF and TNF receptor families contain 8 and 12 members, respectively. The lack of sequence homology among TNF receptor cytoplasmic domains, required for signal transduction, suggests the existence of a specific signaling pathway for each receptor (reviewed in Smith et al. (1994)). Recently, it has been proposed that signal transduction through CD40 and TNF-R2 involved the interaction of their cytoplasmic domain with two cytoplasmic proteins, CD40-bp and TRAF2, respectively (Rothe et al., 1994;Hu et al., 1994). Thus, CD40-bp and TRAF2 could be latent cytoplasmic transcription factors, which would be translocated to the nucleus under receptor activation by their respective ligands. A similar model has already been proposed for the protein family of signal transducers and activators of transcription (STAT) involved in gene activation pathways triggered by interferons (Darnell et al., 1994). This system implies a direct signal transduction pathway through STAT migration from cytoplasm to nucleus, presumably triggered by STAT phosphorylation following receptor activation (Ihle et al., 1994). This is the first report of TNF receptor-associated protein FIG. 8. In situ hybridization of CART1 mRNA in human breast carcinoma and axillary lymph node metastasis. Sections of normal breast (A), in situ carcinoma (C), invasive carcinoma (B), and metastatic lymph node (D) were hybridized with antisense 35 S-RNA probe specific for CART1. CART1 is strongly expressed in the tumoral epithelial cells, whereas the stromal part of the tumor is totally negative (B). CART1 transcripts are homogeneously distributed throughout the positive areas (B-D). Normal ducts are devoid of CART1 signal (A). No significant labeling above background was found when using sense human CART1 RNA probe (data not shown). A-D, bright field micrographs.
FIG. 9. Immunoperoxidase staining of CART1 protein in paraffin-embedded section of invasive breast carcinoma. Sections of human invasive breast carcinoma were stained using hematoxylin (A) and immunostained using a rabbit polyclonal antibody directed against a CART1-specific synthetic peptide (B); the CART1 protein is located in the nucleus of the malignant epithelial cells. A and B, ϫ60 magnification.
FIG. 10. Comparison of CART1, CD40-bp, TRAF2, and DG17 protein structural organization. The size and position of RING finger, CART motif, ␣ helix, and TRAF-C domain are represented for each of these proteins, highlighting the similarity in the organization of these proteins. aa, amino acids. expression in vivo in malignant tissues. In contrast to TRAF2 and CD40-bp, which have been shown to be ubiquitously expressed in normal tissues (Rothe et al., 1994;Hu et al., 1994;Mosialos et al., 1995), no CART1 expression was observed in a panel of normal human tissues including breast, lymph node, heart, brain, skin, lung, stomach, colon, liver, kidney, and placenta. CART1 gene expression is restricted to some primary breast carcinomas and metastatic axillary lymph nodes. CART1 transcripts were specifically detected in malignant epithelial cells and homogeneously distributed throughout the carcinomatous areas. This expression pattern suggests that CART1 could be involved in processes leading to the formation and/or progression of primary carcinomas and metastases. Moreover, immunostaining analysis showed that, in malignant cells, CART1 protein was localized in the cell nucleus. This observation is consistent with the presence of NLS consensus sequences in CART1 and constitutes the first evidence of a possible nuclear function for a member of the TRAF protein family. TNF ligand family members have been shown to induce pleiotropic biological effects, including cell differentiation, proliferation, or death, all processes involved during carcinogenesis and tumor progression (Smith et al. (1994) and references therein). Very little is known concerning the involvement of TNF-related cytokines in breast cancer. TNF-R1 and TNF-R2 have been shown to be expressed in tumoral tissues, and a dramatic increase of the secretion of TNF␣ has been associated with the presence of metastases (Pusztai et al. (1994) and references therein).
From all of these observations, we assume that CART1 may participate in TNF-related cytokine signal transduction pathway, and the nature of protein(s) and/or nucleic acid(s), which may interact with CART1, is now under characterization.