2Breast Cancer Biology Group, Imperial Cancer Research Fund, Guys Hospital, London, U.K., 3Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA, and 4Dipartimento di Patologia Sperimentale, Via S. Giacomo 14, 40126 Bologna, Italy
Received on November 6, 2000; revised on January 12, 2001; accepted on January 16, 2001.
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Abstract |
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Previous reports have indicated a strong correlation between mammary cancers and elevated ST6Gal expression in rats and in human patients. However, we uncovered neither elevated expression of ST6Gal mRNA nor appearance of p4 class in mouse breast carcinomas experimentally induced by transformation with the polyoma-middle T oncogene. A number of established breast carcinoma cell lines were also examined, with ST6Gal mRNA and activity generally low. Moreover, with the exception of the Shionogi cell line, p4 class of ST6Gal mRNA was not expressed in any of the mouse breast carcinoma specimens examined.
Taken together, our data indicate that murine ST6Gal induction during lactation is achieved by de novo recruitment of a normally silent promoter. Furthermore, the data provide no support for elevated Siat1 expression on the mRNA level in association with murine mammary gland carcinogenesis. With the single exception of the Shionogi cell line, the p3 class remains the predominant ST6Gal mRNA expressed in all other murine mammary carcinoma cells examined.
Key words: lactation/mammary gland/mouse/sialyltransferase/ST6Gal
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Introduction |
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During lactation, sialyltransferase enzymatic activity is significantly elevated in the serum of rat (Bushway et al., 1979) and bovine (Sherblom et al., 1986
) but not human (Rajan et al., 1983
). In rat, high sialyltransferase activity in the mammary gland during proliferation and involution accompanies the high activity found in serum (Ip, 1980
). Elevated serum and tumor sialyltransferase activity has been reported in human breast carcinoma (Abecassis et al., 1984
; Dao et al., 1986
), and high ST6Gal levels have been linked to a poor prognosis (Recchi et al., 1998b
). Elevated serum and tumor sialyltransferase activity has also been experimentally reproduced in rat breast carcinomas (Fox et al., 1981
).
We report here that ST6Gal is dramatically induced in the mouse mammary gland during lactation. This ST6Gal mRNA induction is mediated by recruitment of a novel 5'UT exon, exon L, probably driven by a lactogenic promoter, tentatively named P4.
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Results |
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Three other RNA samples of mouse mammary epithelial origin were also subjected to the identical 5'-RACE procedure; the results are summarized in Table I. One of them, an immortalized cell line derived from normal mouse mammary gland (NMG), yielded 17 RACE clones. In striking contrast to RACE clones derived from lactating mammary gland, there were no Exon L forms among these 17 clones. Instead, the vast majority (12) were Exon Ocontaining P3 forms. Among the remaining five clones, four represented N1 forms, and one represented the Exon Hcontaining P1 (hepatic) form. Another RNA sample was from the mouse breast carcinoma line 410.4, from which 14 RACE clones were generated and analyzed. The 5'-RACE profile of 410.4 was similar to that of cells derived from normal mammary epithelium; 12 410.4 clones were Exon Ocontaining P3 forms and the remaining two were Exon L forms. However, the Siat1 mRNA profile of Shionogi (SHI), another mouse breast carcinoma cell line, was similar to that of lactating mammary gland. Eight SHI-derived RACE clones were sequence analyzed; all eight clones represented the Exon L form.
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P4-form of ST6Gal mRNA is restricted to mammary glands during lactation
The data shown up to this point was derived from mouse strain FVB x [FVB(C57xCBA)]. To confirm that the novel P4 (Exon Lcontaining) mRNA form is expressed during lactation in a different mouse strain, the mouse strain CBA was examined. Mammary gland RNA was isolated from virgin, day 14 pregnancy and day 5 postpartum CBA animals. As assessed by a probe against the shared Exon II, there was a striking increase (over fivefold) in overall ST6Gal mRNA levels postpartum (Figure 3A). Mammary gland from day 14 pregnant animals, on the other hand, showed only a modest increase (around twofold) when compared to mammary gland from a virgin animal. When subjected to 5'-RACE analysis, virgin mammary gland expressed only P3 form, commonly regarded as the constitutively expressed ST6Gal mRNA form, and only P4-type (Exon Lcontaining) clones were recovered from mammary gland of day 5 postpartum animals. A mixture of P3 and P4 clones was recovered from 5'-RACE analysis of mammary glands from animals in the 14th day of pregnancy (Figure 3C).
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A Northern blot panel of mouse tissues was used to assess the range of P4-mRNA expression (Figure 4). The level of overall ST6Gal mRNA expression, as measured by a probe against the shared Exon II, varied among tissues examined. The P4 form, as visualized by a probe against Exon L, was present only in lactating mammary gland (Figure 4). In contrast, the constitutively expressed P3 form, as visualized by a probe against Exon Q, was present in all tissues surveyed, including virgin mammary gland (Figure 4).
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Discussion |
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In contrast to a previous report of high levels of ST6Gal mRNA in mouse mammary gland (Takashima et al., 1999), our findings indicate that only a low basal level is present in virgin mammary glands (see Figures 3 and 4). This basal level is maintained by expression of the ubiquitously expressed P3 form. The novel mRNA form is not detectable in virgin mammary gland either by Northern blot or by the more sensitive reverse transcriptase PCR analysis (data not shown). This unique mRNA form, designated as P4 to be consistent with the chronological reporting of the mouse Siat1 forms described to date (Hu et al., 1997
; Wuensch et al., 2000
), differs by a unique 5'-untranslated region derived from an upstream exon, Exon L, in the Siat1 locus. The data is consistent with transcription initiation of the P4-form at the 5' juncture of Exon L (Figure 2). The expression pattern of the P4 mRNA form is consistent with that of a lactogenic-responsive gene. However, it is noteworthy that a STAT5 consensus motif of lactogenic promoters (Groner and Gouilleux, 1995
; Kazansky et al., 1995
; Liu et al., 1995
) is not found within 2 kb of the predicted P4 transcription initiation site (data not shown).
Elevated ST6Gal mRNA has been reported in human breast cancer, particularly in woman with histoprognostic grade III cancer (Recchi et al., 1998a), raising the intriguing possibility of aberrant recruitment of the lactogenic ST6Gal promoter in tumorigenesis. However, in a survey of a number of mammary gland carcinoma cell lines, as well as several virally induced in vivo tumours studied, there was no evidence for an increase in ST6Gal transcripts or for elevated enzyme activity. The rationale for this apparent discrepancy is not clear, although it should be kept in mind that this limited panel of cell lines is unlikely to represent all normal and pathological states of mammary cells. In support of this, only one cell line, SHI, has been found to express the P4 form that predominates in vivo in lactating mammary glands. Moreover, the steady state level of ST6Gal mRNA in SHI is extremely low, measuring only one-tenth that observed in lactating mammary gland, and one-third to one-half that observed in the nonlactating mammary gland line, NMG.
An additional interesting observation arising from the survey of ST6Gal expression is the inconsistent correlation between ST6Gal mRNA level and the measurable enzymatic activity (see Table II). The cell line NMG, for example, exhibits enzymatic activity equivalent to that of N202-neu while maintaining a three- to fourfold lower steady-state ST6Gal mRNA level. Indeed, differential posttranscriptional regulation as a consequence of unique 5'-UTR domains in the mRNA isoforms has been proposed (Aasheim et al., 1993; Dall'Olio et al., 1999
). However, this mechanism is unlikely to participate here because both N202-neu and NMG recruit the P3 form as the predominantly expressed ST6Gal mRNA. Considered together, the data suggest multiple mechanisms are operative in dictating ST6Gal expression in mammary glands.
The functional significance of ST6Gal elevation in the lactating mammary gland can only be speculated at this point. The obvious reason is to address demands for elevated synthesis of SA2,6Galß1,4GlcNAc structure, present both as free oligosaccharide in milk and also as covalent modifications in cellular and milk glycoproteins (Kobata et al., 1969
). Moreover, ST6Gal may also be capable of elaborating the synthesis of SA
2,6GalNAcß1,4GlcNAc-R (Nemansky and Van den Eijnden, 1992
), another structure found in abundance in colostrum (Coddeville et al., 1992
; Nakata et al., 1993
; Girardet et al., 1995
). Moreover, the enzyme itself may be secreted into colostrum (Paulson et al., 1977
) in a manner analogous to enhanced deposition of ST6Gal enzyme from liver into serum during the hepatic inflammatory response (Kaplan et al., 1983
; Jamieson et al., 1993
).
Coincident with elevated ST6Gal expression in mammary gland of lactating animals, high levels of ST6Gal are also present in the intestinal epithelium of newborn animals while nursing, and weaning is concomitant with a conversion of terminal sialylation to fucosylation (Biol et al., 1991; Hamr et al., 1993
; Vertino-Bell et al., 1994
). Although the physiologic significance of these events is far from clear, a tantalizing postulate is a contribution of milk and intestinal epithelial ST6Gal to innate immunity in the newborn animals. Many of the Sia-binding pathogens exhibit a preference for the
2,3-sialyl linkage (Karlsson, 1995
), but the contribution of elevated
2,6-sialyl linkage to innate immunity may be as "decoys" or "smoke screens" to foil potential pathogens (Gagneux and Varki, 1999
).
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Materials and methods |
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Cell lines and tissues
Cell lines NMG and SHI were obtained from central cell services, Imperial Cancer Research Fund. Cell line 410.4 was an established in-house line. All lines were grown in E4/10% fetal calf serum/pen/strep/0.5% Amphotericin B. LM was obtained from a day 9 postpartum FVB x [FVB(C57 x CBA)] mouse, sacrificed, with all four glands removed and snap frozen. CBA mammary gland tissue taken from virgin, day 14 pregnancy, and day 5 postpartum were provided by Clive Dickson, ICRF. Cell lines N202-neu (Nanni et al., 2000), TSA-MC and BFC3 were kindly provided by P. L. Lollini, Department of Experimental Pathology (University of Bologna, Italy). N202-neu cells overexpress neu oncogene, TSA-MC are derived from spontaneous tumorigenesis in Balb/c mice, and BFC3 result from insertional mutagenesis of MMTV. The latter cell line can differentiate in vitro after postconfluent culture.
Mice bearing the MMTV LTR fused to the polyomavirus middle-T oncogene were generated by Guy et al. (1992) on a FVB background. Four heterozygous males were obtained from the Beatson Institute and crossed with Poly-T negative females (C57 x CBA) and progeny screened for Poly-T via PCR. Mammary gland tumors were isolated from Poly-T homozygotic animals and snap frozen.
RNA and enzymatic analysis
For 5'-RACE analysis unless otherwise stated, 1 µg of polyA+ RNA was annealed to the primer mST1-P1 (5'-GATGATGGCAAACAGGAGAA-3') and reverse transcribed. MST1-P1 is complementary to a region in Exon II, such that authentic reverse-transcription events of ST6Gal mRNA must span at least the Exon IExon II boundary. The resultant cDNA was ligated to the marathon adaptor sequence as per instructions and subjected to PCR on a Perkin Elmer thermo-cycler, using the Touchdown program as recommended by Clonetech (Oxford, UK), using the anchor primer AP1 (5'-CCATCCTAATACGACTCACTATAGGGC-3') and the ST6Gal exon I anti-sense primer md11 (5'-CTGCTTCTGGCTAATCTTCTGGGGTTGG-3'). PCR amplification of ST6Gal sequence from contaminating genomic DNA is not possible because ST6Gal gene does not contain sequences that will specifically anneal the anchor primer. Touchdown PCR parameters are 94°C for 1 min, 5 cycles of 94°C for 30 s, 72°C for 4 min, 5 cycles of 94°C for 30 s, 70°C for 4 min, and finally 25 cycles of 94°C for 20 s, 68°C for 4 min. The PCR products were cloned into the plasmid vector pCR2.1 (Invitrogen) and sequenced.
For primer extention/5'-RACE analysis shown in Figure 2, primers mSTl-LP-1 or mSTl-LP-2 were used instead of mST1-P1 for reverse transcription. PCR amplification was achieved using the anchor primer AP1 and either mSTl-LP-1 or mSTl-LP-2. The sequences for mSTl-LP-1 and mSTl-LP-2 are denoted in Figure 2. 5'-RACE products were routinely cloned into pCR2.1. Clones were selected at random and sequence analyzed. For sialyltransferase enzymatic assays, the activity of total cell homogenates toward asialotransferrin was determined in the range of linearity with respect to protein concentration, determined according to the Lowry method as previously described (Dall'Olio et al., 1996).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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