Analysis of the HMGI nuclear proteins in mouse neoplastic cells induced by different procedures

Experimental

Cell Research 184 (1989) 538-545

SHORT NOTE Analysis

of the HMGI Nuclear Proteins in Mouse by Different Procedures

Neoplastic

Cells Induced

VINCENZO GIANCOTTI,*.’ EMANUELE BURATTI,‘” LAURA PERKSSIN,? SONIA ZORZET,? ALAN BALMAIN,* GIUSEPPE PORTELLA,’ ALFRED0 FUSCO,’ and GRAHAM H. GOODWINll *Divartimento di Biochimica, Biqfisica e Chimica delle Macromolecole, Universitd di Trieste, Piazzale Europa I, 34127 Trieste, Italy; tlstituto di Farmacologia e Farmacognosia, Universitci di Trieste, Via Valerio 32, 34127 Trieste, Italy; SThe Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 IBD, Scotland; “Centro di Endocrinologia Sperimentale, Dipartimento di Biologia e Patologia Cellulare e Molecolare, II Facoltci di Medicina e Chirurgia, Universitic di Napoli, Via Pansini 5, 80131 Napoli, Italy; and “Institute of Cancer Research, Chester Beatty Laboratories, Fulham Road, London S W3 6JB, United Kingdom

Four malignant tumors induced in mouse by different experimental procedures were compared as regards their high-mobility-group (HMG) proteins. All tumors showed the complete set of three HMG proteins which we call HMGI-C, I-D, and I-E. The presence of the three HMGI proteins is a characteristic of the transformed phenotype regardless of whether the tumor was chemically, virally, or spontaneously derived. However, the level of expression of the HMGI proteins is not constant in the four tumors. Using reverse-phase HPLC, the individual HMGI proteins were isolated from the spontaneously derived tumor (Lewis lung carcinoma) and shown by amino acid analysis to be similar to those previously obtained from a tumor grown in nude mice by inoculation of in vitro-transformed cells. @ 1989 Academic

Press, Inc

The nuclei of eukaryotic cells contain two families of chromosomal structural proteins which are characterized by their highly polar nature and by their small number of hydrophobic amino acids [ 11. In mammals one of these families is composed of the proteins HMG12 and HMG2, the other of the proteins HMG14 and HMG17. More recently other HMG proteins were identified; this new family has been termed HMGI and has three major components which we call HMGI-C, HMGI-D, and HMGI-E [2]. HMGI-D and HMGI-E correspond to the proteins HMGI and HMGY described by Laland and co-workers [3, 41. All terminally differentiated nondividing cells contain HMG14 and 17, but few or no HMGI proteins. On the other hand, rat cells transformed in vitro with viral oncogenes have high levels of HMGI proteins and low levels of HMG14 and 17, suggesting that neoplastic transformation is accompanied by changes in the ratios of the two ’ To whom reprint requests should be addressed. 2 Abbreviations used: HMG, high-mobility-group proteins; DMB, dimethyl-benzanthracene; TPA, 12Otetradecanoylphorbol acetate; RSB, reticulocyte standard buffer; PMSF, phenylmethylsulfonyl fluoride; PCA, perchloric acid; SDS, sodium dodecyl sulfate; HPLC, high-performance liquid chromatography; TFA, trifluoroacetic acid. Copyright @ 1989 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827189 $03.00

538

Short note

539

families of proteins [5, 61. The high levels of some HMGI proteins are also characteristic of undifferentiated proliferating cells [6, 71. In this paper we report a study of the ubiquity of the HMGI proteins in malignant tumors induced by different experimental protocols. For this reason we selected three models of in uiuo carcinogenesis in which neoplastic transformation rose from completely different types of induction. The first system was a mouse skin carcinoma induced by treatment with the initiating agent dimethylbenzanthracene (DMBA) followed by treatment with the promoting agent 12-O tetradecanoylphorbol-acetate (TPA) according to Refs. [8-l 11. The second system consisted of the lung metastasis which appears in the lungs of athymic mice as a consequence of primary thyroid tumors induced by intrathyroidal injection of the Kirsten murine sarcoma virus [ 121.The third system was the Lewis lung carcinoma which is a tumor which arose spontaneously in the lungs of a C57BL/6 mouse [13] and has been maintained in uiuo by serial transplantations [14]. Data on nuclear proteins from these three models of carcinogenesis were compared with those obtained from a tumor arising from the injection in nude mice of cells transformed in vitro with the c-myc proto-oncogene and polyoma middle T gene. We called this tumor PC-C13-myc-PyMuLV [6] and a sample of nuclear proteins extracted from it was used as reference in this research. In this report we demonstrate that all three tumors contain high levels of HMGI proteins as shown previously in the cases of the cells transformed in vitro and of the tumor derived from them [5-71. Therefore we draw the conclusion that the presence of the complete set of the three HMG proteins I-C, I-D, and I-E is a constant feature of highly malignant mouse tumors regardless of whether they were chemically, virally, or spontaneously originated. Moreover we concentrated our attention on Lewis lung carcinoma because of its pharmacological interest [15, 161. This tumor was also used as a suitable source for purification of large quantities of the three HMGI proteins using reverse-phase chromatography. We obtained samples of HMGI proteins whose amino acid analyses correspond to those of the proteins prepared from PC-C13myc-PyMuLV cells [6]. However, the levels of expression of some HMGI proteins are different in the two tumors. Materials and Methods PC-C13-myc-PyMuLV tumors were grown in nude mice using in uirro-transformed cells as previously reported [6]. Chemically induced skin carcinomas were obtained in mouse by the combined treatment of DMBA and TPA [g-l 11. Virally induced tumors were grown in Fischer rat thyroids by intrathyroidal injection of the Kirsten sarcoma virus. In almost 30% of the virally treated animals, lung metastases appeared as white nodules 2 months after viral treatment [ 121. The Lewis lung carcinoma line was originally provided by the National Cancer Institute (U.S.A.) and was maintained in C57BL/6 mice by subcutaneous implantation of lo6 single viable tumor cells in the axillary region. From this tumor a single suspension was prepared, and inoculation in BD2Fl mice to obtain the tumors used for this research was performed as previously reported [14J. Nuclei from Lewis lung carcinoma were prepared essentially according to the method reported in a preceding paper [17]. Tumors were suspended in hypotonic reticulocyte standard buffer (RSB; 10 mM NaCI, 3 mM MgQ, 10 mM This, pH 7.4) and homogenized on ice in a blender for 15 min. The suspension was filtered through gauze and centrifugated at 1,500g. The pellet was resuspended in

540 Short note +A.

I-E

c

UREA

I-D

D

Fig. I. Two-dimensional gel electrophoresis of perchloric acid extracts of (A) mouse skin carcinoma, (II) rat lung metastasis, (0 Lewis lung carcinoma, and (D) PC-Cl3-myc-PyMuLV tumor, showing HMG14 and 17 and the three HMGI proteins, I-C, I-D, and I-E.

RSB containing 0.5% of Nonidet-P40 and homogenized in a glass Dounce homogenizer (pestle S). Nuclei were recovered by centrifugation and washed with RSB without Nonidet. To inhibit proteases, all procedures were carried out in the presence of 0.5 mM phenylmethylsulfonyl fluoride (PMSF). HMG and histone Hl proteins were selectively extracted from the tumors or nuclei with 5% perchloric acid (PCA) and acetone-precipitated as described previously [5]. Two-dimensional electrophoretic analysis was carried out in the first dimension by acetic acid-urea polyacrylamide gel electrophoresis followed by SDS electrophoresis in the second dimension [S]. Reverse-phase HPLC was carried out as previously described [6,7] using a BioRad RP-304 column (250X21.5 mm) for large-scale fractions (20 mg) or a RP-304 (250x4.6 mm) for small-scale (about I mg) fractionations.

Results In a previous report we showed that rat thyroid epithelial cells transformed by a variety of oncogenes or by a combination of the c-myc proto-oncogene and the polyoma middle T gene exhibited high levels of three nuclear phosphoproteins related to the HMG protein family. These three proteins, which we term in this paper HMGI-C, HMGI-D, and HMGI-E, are present at much lower levels in normal untransformed cells growing in culture and are undetectable in fully differentiated nondividing tissues [5-71. To analyze the HMGI proteins in tumors induced by other treatments, total HMG proteins plus histone Hl were extracted with perchloric acid from (A) a chemically induced mouse skin carcinoma, (B) a rat lung metastasis appearing after intrathyroidal injection of the Kirsten sarcoma virus (which harbors the Kirsten ras oncogene), and (C) the spontaneously derived Lewis lung carcinoma tumor grown in mice. The extracted proteins were resolved by two-dimensional gel electrophoresis and compared with the previously described HMGI proteins from thyroid cells transformed with c-myc and polyoma middle T genes (termed PC-C13-myc-PyMuLV cells). Figure 1 shows the relevant region of the twodimensional gels on loading equal amounts of PCA-extractable protein from the

Short note

541

I-D

8B

6-

B

-60

‘4-

8642JI I 30 300 320 340 360 380 ELUTION VOLUME (mL)

I

400

Fig. 2. Reverse-phase HPLC fractions of 20 mg perchloric acid-extracted protein from (A) PC-C13myc-PyMuLV tumor and (B) Lewis lung carcinoma. Only the first half of the elution profiles is shown where the HMG14/17 and HMGI elute. Solvent A: 0.1% TFA in water; solvent B: 5 % water/95 % acetonitrile (v/v) treated with 0.1% TFA.

four tumors. In all four cases the levels of HMGI-D and HMGI-E are very much higher than those of HMG14 and 17. HMGI-C is present in variable amounts in the four tumors. These results, therefore, show that the high levels of HMGI-D and HMGI-E and the presence of the HMGI-C are a general feature of neoplastic cells whether they are transformed by viral oncogenes or carcinogens. In the case of the Lewis lung carcinoma, the transforming agent is unknown. Lewis lung carcinoma was selected for further biochemical analysis since it is a malignant tumor widely used as an experimental model in biological studies and it produces a systemic neoplastic disease similar to human carcinoma because of its surgical incurability and low response to antitumor agents [15, 161. A highresolution analysis of HMGI proteins was afforded by reverse-phase HPLC. Figure 2 shows the elution profile of proteins from Lewis lung carcinoma in comparison with the previously described elution profile of HMGI proteins from PC-C13-myc-PyMuLV tumor [6]. Figure 3 shows the electrophoretic analysis of the eluted proteins of interest. The elution profiles of the HMG proteins from the two cell types are very similar, showing the higher levels of HMGI-D and I-E relative to HMG14 and 17. The level of expression of the protein I-C is different in the two tumors as is seen in the two-dimensional electrophoretic analysis of

542 Short note TABLE 1 Amino acid composition (% mol) of HPLC fractions and rat thymus HMG14 and 17 Lewis lung carcinoma

Asx Thr Ser Glx Pro GUY Ala Val Met Ile Leu Tyr Phe His LYS Arg

Rat thymus

I-E,

I-E*

I-C

I-D,

I-D2

HMG14

HMG17

3.1 6.8 9.8 18.0 14.7 12.7 7.7 2.9 0.1 1.1 2.5 0.1 0.2 0.0 10.0 10.2

2.0 5.8 9.3 17.2 11.9 13.6 6.1 2.9 0.0 0.0 2.8 0.0 0.0 0.0 16.7 11.7

5.4 7.1 10.0 16.0 12.8 12.1 10.7 3.9 0.0 1.1 1.6 0.4 0.4 0.2 12.5 5.7

4.4 5.4 10.7 19.1 11.0 10.0 6.7 3.7 0.5 0.9 2.7 0.0 0.0 0.0 15.9 9.0

9.0 4.8 8.2 26.2 8.5 10.0 6.5 3.8 0.0 0.7 2.0 0.0 0.0 0.0 12.8 7.1

7.8 4.6 8.2 16.4 7.3 8.1 13.2 3.2 0.1 1.1 2.3 0.0 0.0 0.2 20.9 7.8

13.7 1.8 2.8 9.0 10.0 9.3 20.2 2.6 0.0 1.1 0.0 0.0 0.0 0.0 25.8 4.1

Fig. 1. HMGI-E splits into two peaks I-Et and I-Ez. Heterogeneity is also observed in the HMGI-C and HMGI-D peaks. Rechromatography of HMGI-D results in the partial separation into two components I-D, and I-D1 (not shown). The amino acid composition of HMGI proteins isolated from Lewis lung carcinoma is reported in Table 1 together with the composition of HMG14 and 17 from rat thymus as reference [7]. Amino acid composition confirms that the assignation of the chromatographic peaks as HMG proteins is correct. In comparison with lung carcinoma, normal rat tissue has low levels of the three HMGI proteins as shown in the chromatographic profile of Fig. 4 A. HMGIC is not detectable, at least at these protein loadings. An indication of the relative levels of the proteins in normal and carcinoma lung was obtained by measuring the peak areas of the HMGI-D and HMGI-E proteins and histone Hl (not shown in the figure) in the elution profiles. The HMGI-D/H1 and the HMGI-E/HI ratios were approximately lo- to 15fold higher in lung carcinoma than those in normal lung. Since the Hi/DNA ratio of different cell types is constant, it would appear that in Lewis lung carcinoma there is an elevated expression of HMGI proteins. Both chromatographic profiles of Figs. 3 and 4 show a peak (B) eluting after HMGI-D. Preliminary data (not shown) suggest this peak contains a protein that could be related to a nucleolar protein named nucleolin [ 181.Finally, HMGI were demonstrated to be nuclear proteins by isolating nuclei from Lewis lung carcinoma and analyzing the PCA extract by HPLC (see Fig. 4 C).

Short note 543 12345678

HlB-

--B

I-C{

7%

-1-D

I-E-

17-

Fig. 3. Acid-urea polyacrylamide gel electrophoresis of the Lewis lung carcinoma HMG proteins purified by the reverse-phase HPLC shown in Fig. 2B. (1) Total Lewis lung carcinoma PCA-extracted protein; (2) HMG17 peak; (3) HMGI-E, peak; (4) HMGI-E2; (5) HMGl4 peak; (6) HMGI-C peak; (7) HMGI-D peak; (8) B peak. (It should be noted that the band labeled HMG14 in lane 1 of the total PCA-extracted material is in fact a mixture of proteins most of which migrate at the front in seconddimension SDS gel while only a small proportion migrates in HMG14 position.)

Discussion In this paper we report a study on the HMG proteins which are present in mouse tumors obtained by different experimental procedures. We demonstrated that the neoplastic transformation in uiuo is always accompanied by the appearance of three proteins (I-C, I-D, and I-E) which constitute the HMGI family. HMGI-D and HMGI-E are always the major components in comparison with both HMG14 and 17 (which are present in all mammalian and avian cells) and the presence of HMGI-C protein also seems to be a characteristic of neoplastic cells. In preceding studies carried out in vitro [5-71, we demonstrated that proteins I-D and I-E are also present in some untransformed but immortalized lines as fibroblasts, while I-C appears following full transformation with oncoviruses. In this study we report that neoplasms obtained by transformation in uiuo exhibit the protein I-C together with elevated levels of proteins I-D and I-E. The presence of the complete set of the three proteins is independent of the experimental procedure used to obtain the tumors, suggesting that it is a peculiar feature of the transformed phenotype. A detailed biochemical analysis of the HMG proteins of the Lewis lung carcinoma using HPLC and amino acid analyses demonstrated that this spontaneously derived tumor contains the same HMG pattern as that found in the tumor 35-898340

544 Short note I

1

I

a-

14

I

I-D

1

B

6 -c 4-

1-E 1

2 -17

\

J

E 2

I

I

I

I

: l-6-B a

34x b7

SJ-

Fig. 4. Reverse-phase HPLC fractions of the PCA-extracted proteins from (A) normal lung tissue from mouse, (B) Lewis lung carcinoma (total tissue), and (C) purified nuclei prepared from Lewis lung carcinoma. Approximately 0.8 mg of total protein was loaded onto the column in each case.

obtained by injection into nude mice of cells transformed in vitro. Lewis lung carcinoma shows a typical profile of HMG proteins characteristic of transformed rat or mouse cells, namely high levels of HMGI proteins and depressed HMG 14 and 17. All three HMGI proteins are always present as multiple forms whose origin has been unknown until now. The HMG 14 and 17 proteins have been sequenced and their genes isolated and extensively analyzed 11, 21. Two of the HMG proteins, HMGI and HMGY, in the nomenclature of Laland et al. [3, 41, have been sequenced and have recently been cloned 14, 191.HMGY corresponds to one or both of the HMGI-E proteins described by us and HMGI corresponds to one of the HMGI-D proteins. HMGY and HMGI appear to be products of differential splicing of transcripts from a common gene(s) (R. Reeves, manuscript submitted). HMGY (HMGI-E) protein differs from HMGI (HMGI-D) in having a deletion of an 11 amino acid peptide present in the N-terminal of HMGI. Thus the HMGI-D and I-E chromatographic subfractions described in this paper could be

Short note

545

splicing variants from one or more genes or could be due to postsynthetic modifications. HMGI-C is currently being sequenced in our laboratories, but its relationship to the other HMGI proteins is not yet known. The HMG 14/17 family is supposedly associated with transcriptionally active genes (although this is by no means certain [2]), while the HMGI proteins are probably associated with AT-rich sequences in centromers, telomers, and G/Q bands of metaphase chromosomes (R. Reeves, manuscript submitted). The HMGI proteins are, therefore, likely to be involved in the condensation of these chromosomal regions during mitosis. The question is, why should transformed cells exhibit high levels of these proteins? It is conceivable that in normal dividing cells the proteins are synthesized just prior to mitosis to condensed telomers and centromeres, but in transformed cells the HMGI genes may be constitutively transcribed throughout the cell cycle, hence the higher overall levels in a mixed population of cells. The authors thank Dr. R. Reeves for making available preprints on HMGI genes and immunolocalization. This research was funded by grants from Consiglio Nazionale delle Ricerche (CNR), Rome; Minister0 della Pubblica Istruzione, Rome; Associazione Italiana per la Ricerca sul Cancro, Milan; Progetto Finalizzato Oncologia de1 CNR, Rome; and the Cancer Research Campaign and Medical Research Council, United Kingdom.

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