(CANCER Ri:Si:\ROI 49. 6949-6952. December 15. 1989] Changes in Calcitonin Gene RNA Processing during Growth of a Human Medullary Thyroid Carcinoma Cell Line1 Barry D. Nelkin,2 Kurt Y. Chen, AndréedeBustros, Bernard A. Roos, and Stephen B. Baylin The Oncology Center, Johns Hopkins I'niversity School of Medii -inc. Baltimore, Maryland 21205 /B. I). ,V.. K. Y. C., A. de B., S. H. H./; the (ieriatric Research. Education, ami Clinical Center (dRECC). I 'eterans Administration Medical Center, Tacoma. H'ashington 98493 /B. A. R./: and the I 'nirersily of Hushinnton, Seattle, Washington VXIV? [R. A. R.I ABSTRACT sive product (2). To date, the factors influencing the posttran scriptional choice of CT or CGRP mRNA remain unknown. The ratios of calcitonin (CT) to calcitonin gene-related peptide (CGRP) We have studied CT gene regulation in the TT cell line, mRNA, both generated by alternative RNA processing from the same which was derived from a rapidly growing human MTC (7) and primary RNA transcript, arc shown by Northern blotting of cytoplasmic RNA to vary as a function of growth in a human medullary thyroid produces high levels of both CT and CGRP. We have previously carcinoma cell line ('IT). I'pon initial seeding, CT mRNA levels are shown that, in this cell line, levels of the CT peptide are relatively high, and CGRP mRNA levels are relatively low. During the modulated dynamically during various stages of growth in early logarithmic growth phase, CGRP mRNA levels rise severalfold, culture, being lowest during times of rapid cell proliferation while CT mRNA levels change only slightly. As the cells approach and highest in cells at saturation density (8). In vivo, it has also confluence, both CT and CGRP mRNA levels rise. Subsequently, CGRP been found that CT is decreased in MTC only from patients mRNA levels fall substantially in postconfluent cells, while CT mRNA with rapidly growing, aggressive tumors. In order to determine levels remain high. By actinomycin I) blocking of nascent transcription, the dynamics of CT gene expression which may regulate this we have shown that these growth-related, reversible changes in the ratio modulation of CT production, we have examined, using several of CT to CGRP mRNA arc not due to changes in mRNA stability. Our data rather suggest that 'IT cells reversibly alter alternative RNA- cDNA probes (4), CT gene-derived RNA production during processing patterns dependent upon growth conditions in vitro, such that growth of MTC cells in culture. We now find that the basis for (I mRNA is lowest and CGRP mRNA is highest during rapid growth. this difference is largely at the RNA level and that CT gene The mechanisms underlying this RNA-processing alteration may play a expression bears a complex and dynamically regulated relation role in certain patients with aggressive forms of medullary thyroid ship to cell growth. This regulation includes reversible changes carcinoma, in whom a decrease or loss of CT levels heralds a poor in posttranscriptional processing which lead to changing ratios prognosis. of the relative levels of CGRP to CT mRNAs and in part involves a reversible switch in choice of RNA processing to CT INTRODUCTION or CGRP mRNA. MTC1 is an important experimental system, biologically and clinically, for studies of both peptide hormone gene expression MATERIALS AND METHODS and tumor differentiation and progression. Both the tumor and Cell Culture. TT cells were seeded on day 0 at 1 x IO6cells/25 cm2 its normal counterpart, the thyroid C-cell, express the calcitonin flask, taking care to achieve a single cell suspension, and grown in gene. RPMI 1640 with 16% heat-inactivated fetal bovine serum (Gibco), 2 In both rat and human, the CT gene is composed of six exons HIMglutamine, 100 units/ml penicillin, and 100 i<u ml streptomycin, (Fig. 1) (1-3). By alternative processing of a single primary as described previously (8). The medium was changed on day 6. Under RNA transcript, two separate mature mRNAs derive from this these conditions, the cells have a doubling time of 83 h. In the experi one gene. The mRNA for CT itself contains sequences from ment shown in Fig. 4, TT cells were seeded as above, but serum was exons 1-4, and the mRNA for CGRP contains sequences from reduced to 10% and the medium was changed every 2 days. exons 1-3 and 5-6; during processing of the initial RNA RNA Hybridization and Electrophorcsis. Cells were harvested after various times, and cytoplasmic RNA was extracted using the vanadyl transcript to generate CGRP mRNA, exon 4 is treated as part ribonucleoside complex method (9). Two ug of total cytoplasmic RNA of an intron and is removed. The choice of mRNA produced, from each time point were denatured, electrophorcsed on 1.5% form made at the posttranscriptional RNA-processing level, varies aldehyde gels, and blotted to nitrocellulose (10). Blots were probed with specifically among normal tissues. Thus, normal thyroid C-cells human CT (exon 4)-specific cDNA clone pTT1062. a CGRP-specific predominantly produce CT mRNA, while many cells of the (exons 5 and 6) Äanll restriction fragment of cDNA clone pTT42, or a "common region" (sequences present in both CT and CGRP mRNAs; central nervous system and some other neuroendocrine cells predominantly produce CGRP (3). In the rat, transplantable see Fig. 1(-specific (exons 2 + 3) specific fragment of pTT42 (4). A medullary thyroid carcinoma can differ from normal C-cells in Pvu\\-Pst\ CGRP-1 (11(-specific fragment of pTT42, containing exon 6 and 17 base pairs of exon 5, gave the same results as the ßanll the mode of posttranscriptional processing of the CT gene, and fragment. Autoradiographic density was measured on a Clifford Den- an increase in CGRP mRNA can correlate with a decrease in sicomp densitometer. Loading of RNA was normali/ed by rehybridizing CT mRNA; in some cases, CGRP mRNA is almost the exclu- filters with a /i-actin cDNA probe (pAl, provided by D. Cleveland, Receivcd 2/12/88: revised 6/5/89; accepted 8/24/89. Johns Hopkins University School of Medicine). The actual fraction of The costs of publication of this article were defrayed in part by the payment the total CT gene derived mRNA expressed as CT or CGRP mRNA of page charges. This article must therefore be hereby marked advertisement in was estimated by |-x-32P|ATP end labeling or reverse transcription of accordance with 18 tJ.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by grants from the American Cancer Society RNA extracted from TT cells at various time points and hybridization to CT- or CGRP-specific plasmid DNA sequences immobili/cd on (PDT-207 and NP-533): the American Cancer Society. Maryland Division. Inc.: the NIH (AM .16116); and the Veterans Administration: and by gifts from the nitrocellulose (12) (data not shown). W. W. Smith Foundation and the Hodson Trust. CT and CGRP mRNA Half-Life Measurements. TT cells were seeded 2To whom requests for reprints should be addressed, at the Oncology Center as described above. On days 3, 7, or 11, actinomycin D (8 /¿g/ml)was Research Laboratories. Johns Hopkins University School of Medicine. 424 N. added to block transcription, and cells were harvested after various Bond Street. Baltimore, MD 21231. 1The abbreviations used are: MTC. medullary thyroid carcinoma; CT. calci further incubation times. This method was chosen instead of pulse- tonin: CGRP. calcitonin gene-related peptide; cDNA, complementary DNA. chase techniques (13) since the high concentration of pyrimidines 6949 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. CT RNA PROCESSING CHANGES IN CELL GROWTH CGRP CGRP Common_nLJiCommon2n~Lr~ CT Coding Codmq Untronsloted A Day iRNACTr—LProcessing 79 CT VGRP mRNA Common Region Specil'cCD Probe )CGRP Specific Probe ]CT Spec.lie CGRP Probe Fig. I. Overview of human calcitonin gene structure and expression. Alter native RNA processing produces two mRNAs. one encoding CT [1.0 kilobase (AA)|and one encoding CGRP [1.1 kilobases). Specific common region (exons 2 and 3). CT (exon 4), and CGRP (exons 5 and 6) hybridization probes shown were derived from cloned cDNAs (4). Data from Refs. 1 and 4-6. required for pulse-chase are toxic to TT cells. RNA was extracted as described above, and 2 ¿tgwereapplied to nitrocellulose using a slot blot manifold (Schleicher and Schuell, Keene, NH). Slot blots were hybridized with the CT- and CGRP-specific probes described above. RESULTS We have examined, by Northern blotting using CT- and CGRP-specific mRNA probes, the dynamic variation of the relative levels of these mRNAs in TT cells during growth. Fig. 2, A and B, shows that, during TT cell growth, CT-specific mRNA levels generally paralleled those of total CT gene- derived mRNA (Fig. 2Ä);these relative mRNA levels remained constant or decreased for several days after cell seeding and then gradually increased as the cells progressed toward conflu ence (after day 6). These changes in CT mRNA levels were consistent with our previous observation that CT peptide levels fall during the initial growth phase of the TT cells and increase to initial levels at confluence (8). Thus, CT peptide production in these cells during growth appears to be largely regulated at the mRNA level. When the hybridization was repeated using a CGRP-specific probe (exons 5 and 6), a different pattern emerged. It became apparent that the relative levels of CGRP mRNA varied during growth, in a pattern unlike that of CT-specific or overall CT gene-derived mRNA. Thus, for the first 2-3 days after the cells Fig.