Klinische Wochenschrift
Klin. Wochenschr. 57, 257-265 (1979)
'9 Springer-Verlag 1979
Originalien Comparative Studies on Phenol-Soluble Nonhistone Chromatin Proteins in Normal and Leukaemic Human Leukocytes* S. Seeber, T. Meshkov**, K.P. Brucksch, J. K/iding, C.G. Schmidt and H. Busch Inhere Universitfitsklinik und Poliklinik (Tumorforschung), Westdeutsches Tumorzentrum, Essen Department of Pharmacology, Baylor College of Medicine, Houston, TX, USA
Vergleichende Untersuehungen fiber phenoMOsliehe Chromatinproteine in normalen und leuk~imischen menschlichen Leukozyten Zusammenfassung. Phenol-16sliche Chromatinproteine wurden aus Zellkernen normaler und leukfimischer menschlicher Leukozyten isoliert. Die Proteine, deren Bedeutung im Rahmen der Kontrolle des Transkriptionsvorganges diskutiert wird, wurden durch Analyse ihrer geMektrophoretischen Verteilungsmuster und ihrer Markierung mit 14C-Leuzin oder 32p-Orthophosphat verglichen. AuBerdem wurde der Einflul3 interkalierender Agentien wie Adriamycin untersucht. Die Fraktion phenol-16slicher Nicht-HistonProteine des Chromatins menschlicher Leukozyten enth/ilt mindestens 20-35 individuelle Proteine, welche mit zunehmender Laufgeschwindigkeit auf 10%Polyacrylamidgelen numeriert wurden. Zwischen normalen Lymphozyten und normalen Granulozyten ergaben sich charakteristische Unterschiede mit jeweils einer spezifischen Proteinbande (Nr. 26a bei Lymphozyten; Nr. 25 bei Granulozyten) ftir beide Zelltypen. Interessanterweise war das Protein Nr. 25 in Myelozyten (CML) nicht vorhanden, jedoch in reifen Granulozyten desselben Patienten (CML) nachweisbar. Die radioaktiven Markierungen der Proteine mit 32p (Phosphorilierung) bzw. 14C-Leuzin (Umsatz) nahmen ganz allgemein mit zunehmender Zelldifferenzierung ab. Leuk/imische Lymphozyten unterschieden sich yon normalen Lymphozyten durch erh6hte Protein-Konzentrationen im hochmolekularen Bereich. Beim Vergleich von Zellen aus AML, ALL * These studies were supported by grant Se 161/6 from Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg ** Supported by Humboldt-Stiftung Offprint requests to: Priv.-Doz. Dr. S. Seeber (address see p. 265)
und der CML-Blastenkrise ergaben sich bei weitgehend fihnlichen Mustern keine typischen Differenzen. Ebenso waren gemeinsame leukfimiespezifische Abweichungen gegenfiber Normalzellen nicht nachzuweisen. Lymphosarkomzellen zeigten allerdings quantitative und qualitative Abweichungen vom gew6hnlichen Verteilungsmuster der Zellkernproteine. Nach Einwirkung von Adriamycin in vitro kam es bei den verschiedensten Zellinien dosisabhfingig zu einer selektiven Abnahme eines bestimmten Proteins (Nr. 30) dessen m6gliche Funktion als ,,marker" fiir den Interkatationslocus von Adriamycin an der DNA diskutiert wird.
Sehliisseiwiirter: Phenol-16sliche Chromatinproteinemenschliche Leukfimiezellen - Adriamycin. Summary. Phenol-soluble chromatin proteins which may be involved in gene control mechanisms have been isolated from citric acid nuclei of normal and leukaemic human leukocytes derived from freshly obtained venous blood. They were compared by onedimensional gel electrophoresis and by comparative labelling with ~4C-leucine or 32p-orthophosphate. In addition, the influence of intercalating agents such as adriamycin and daunomycin was studied. Between 20-35 individual proteins were found in the phenol-soluble nonhistone protein fraction of human leukocytes. They were numbered with increasing mobility on 10% polyacrylamide gels. Distinct differences were found between normal lymphocytes and normal granulocytes, with one specific protein (no. 26a in lymphocytes; no. 25 in granulocytes) for both cell types. Interestingly, protein no. 25 was not present in CML myelocytes but in CML granulocytes. 32p_ and leucine labels were generally found decreased
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S. Seeber et al. : Nonhistone Chromatin Proteins in Normal and LeukaemicHuman Leukocytes
with increasing cell differentiation. Leukaemic lymphocytes differed from normal lymphocytes by increased protein concentrations in the high molecular weight region. In comparisons of A M L and ALL cells, including blast cells of C M L blast crisis, no constant differences nor any markers common to leukaemic cells as compared to normal cells were detectable. However, lymphosarcoma cells showed quantitiative and qualitative aberrations from the usual leukaemia pattern. After in vitro incubations of cells with adriamycin a selective decrease of an individual protein (no. 30) was noted. This protein may serve as a marker for the intercalation site.
Key words: Phenol-soluble chromatin proteins - Human leukaemia cells - Adriamycin.
Only few definitive studies have been reported on nonhistone chromatin proteins in human leukocytes [5, 8, 9, 12, 14, 39, 40, 44]. Analyses of nuclear acidic proteins in various human normal and leukaemic cell lines gain importance since it has been shown that some of these proteins are specific in different tissues and in different species [15, 25, 38, 41, 42]. It has been derived from transcriptional studies that nonhistone proteins are probably involved in positive control mechanisms for gene expression [2, 7, 13, 18, 22, 31, 35]. More recently, it has been suggested that differences exist in nonhistone proteins between chromatin of normal cells and tumour cells that may be related to altered gene expression [1, 3, 5, 6, 44]. Earlier studies have shown that chromatin from human leukaemic lymphocytes differed from that of normal lymphocytes by a decreased actinomycin D binding capacity [19, 24] and by a higher template activity [26]. In addition, histone deacetylase activity has been shown to be greater in lymphocytes from leukaemic patients, and the protein responsible for this activity was shown to bind intimately to isolated chromatin [8]. Very recently, biophysical and biochemical characteristics of isolated chromatin and individual chromosomal components of cultured human lymphocytes from normal donors and from patients with infectious mononucleosis and acute lymphoblastic leukaemia were presented [9]. Lymphocyte chromatin had been isolated following the method of Marushige and Bonnet (1966), using homogenization of cells in saline-EDTA buffer and in 0.01 M Tris buffer (pH 8.0) followed by purification of chromatin by centrifugation through 1.7 M sucrose (0.01 M Tris, pH 8.0). These studies showed that a) after thermal
denaturation higher Tm values were found in the leukaemic chromatin, b) differences were present between leukaemic and normal lymphocytes in the ratios o f D N A : RNA: protein, and c) the ratio of nonhistone proteins to histone proteins associated with chromatin was higher in the leukaemic cell lines [9]. In this comparison, amino-acid compositions of the histones were very similar, but the nonhistone chromosomal proteins appeared to be more heterogeneous with respect to amino-acid composition. Also, onedimensional densitometer tracings had revealed considerable heterogeneity when different kinds of human lymphocytic cells were compared [9]. Chromosomal acidic proteins isolated from the purified chromatin of human leukaemic cells greatly stimulated the template activity of the chromatin in in vitro synthesis [9]. Our own studies were begun in order to compare nonhistone chromatin protein patterns from various human leukaemia cells and normal teukocytes which were in all cases freshly obtained from untreated patients. This seemed to be important since after longterm culture the leukaemic nature of cultured leukocytes may be questionable. The goal was to define possible chromatin markers for individual leukaemic cell lines and to search for specific differences between myelocytic and lymphocytic cells which are, at the blast level, often not distinguishable by morphological criteria.
Materials and Methods 1. Separation of Leukocytes fmm Erythrocytes
Freshly drawn heparinized blood was diluted 1:1 (v/v) with neoPlasmagel (3% gelatin, tool. wt 35,000, Na + 142mval/L, Ca ++ 2.8 mval/L, C1- 109mval/L; B. Braun AG, Melsungen, F.R.G.). The mixture was allowed to settle in a siliconized glass cylinder at 37° C for 30-40 min. After sedimentation, both the supernatant and the buffy coat were carefully removed with a Pasteur pipette. The cells were centrifuged for 10 min at 1,000rpm, RC3 Sorvall at 4° C and washed 2 to 3 times with NKM buffer (0.13 M NaC1, 0.005 M KC1, 0.008 M Mg C12). 2. Separation of Mononuclear Cellsfrom Granulocytes
In a one-stage procedure as originally designed by Boyum (1968), separations were carried out with a sodium metrizoate-Ficollmixture (Lymphoprep,density 1.077_+0.001g/ml, 9.6% (w/v) sodium metrizoate, 5.6% (w/v) Ficoll; Pharmacia, Uppsala, Sweden). 3. Isolation of Leukocyte Nuclei with Intact Macromolecutes
A modified citric acid procedure similar to that first described by Dounce (1955) was employed. In studies on nuclear proteins it had been demonstrated that some chromatin proteins were re-
S. Seeber et al. : Nonhistone Chromatin Proteins in Normal and Leukaemic tluman Leukocytes leased from the nucleus when citric acid acid concentrations between 1.5 and 5% were used [34]. These studies had shown that with a dilution of 0.025 M citric acid no visible alterations of the nuclear morphoIogy were detectable and only few, if any, chromatin nonhistone proteins were released selectively, as assayed by the 0.4 N sulfuric acid extract. In addition, the chances for proteolytic degradation during the procedure of nuclear isolation were reduced at the low pH (2.5) maintained during all steps of preparation [44]. Other studies using human leukaemia cells had pointed out the serious degradation problem [40]. NKM-buffer washed leukocytes were suspended in 10 volumes (w/v) of 0.025 M citric acid (pH 2.5) and disrupted with a Super Dispax Model SDT-182 in twelve (lymphocytic cells, myeloblasts) to eighteen (myelocytes, granulocytes) 15s bursts (3 to 4~/2 min of disruption) at a Regler setting of 7 [16, 34]. Crude nuclei were purified through 0.88 M sucrose containing 3.3 mM Ca 2+ and 100 gM PMSF (Sigma Chemical Corporation, St. Louis, Mo., U.S.A.). The nuclear pellet was washed in a buffer containing 0.15 m NaCI, 100 gM PMSF and 0.01 M Tris, pH 7.2 and frozen (Nood6n et al., 1973).
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Results 1. Tissue-SpecS'city o f Phenol-Soluble Chromatin Proteins F r o m e a r l i e r s t u d i e s o f T e n g e t al. ( 1 9 7 1 ) c o m p a r i n g p h e n o l - s o l u b l e c h r o m a t i n p r o t e i n s i n r a t liver, k i d n e y , s p l e e n a n d b r a i n , it h a d b e e n c o n c l u d e d t h a t m a n y of the proteins isolated were tissue-specific. In a control experiment for studies in human leukocytes, citric a c i d n u c l e i w e r e p r e p a r e d f r o m r a t liver, k i d n e y , spleen and brain, and phenol-soluble chromatin p r o t e i n s w e r e i s o l a t e d . A s d e m o n s t r a t e d i n Fig. 1, characteristic differences were noted in the quantita-
4. Isolation of Phenol-Soluble Chromatin Proteins .from Human Leukocytes Purified nuclear pellets from leukaemia cells and normal leukocytes were extracted two times with 0.14 M NaC1. Following centrifugation for 5 min at 3,000 g for 5 rain, the extraction was repeated. The nuclear residue was suspended in 10 volumes of 1:1 (v/v) chloroform-methanol containing 0.2 N HC1. After centrifugation at 5,000 g for 5 min, the sediment was extracted with 10 volumes of 2:1 chloroform-methanol containing 0.2 N HC1 and centrifuged as before. The pellet was washed with ether, resuspended in 5 volumes of a buffer containing 0.1 M Tris-HC1, 0.01 M EDTA and 0.14 M 2-mercaptoethanot, pH 8.4. An equal volume of buffersaturated cold phenol was added and the suspension was allowed to stand for 14 h at 2 ° C. After centrifugation of this mixture (10 rain, 12,000 g), the aqueous phase was re-extracted with an equal volume of phenol and centrifuged as before. The combined phenol phases were dialysed in a three-step procedure that followed exactly the conditions described by Vifiuela et al. (1967) and Teng et al. (197I). The phenol-soluble proteins were thus restored to the aqueous phase and ready for further characterization.
5. Separation of Phenol-Soluble Chromatin Proteins by Analytical One-Dimensional Gel Electrophoresis Partial resolution of the complex mixture of phenol-soluble chromatin proteins was achieved on 0.6 x 14 cm analytical gels composed of 10% acrylamide, 0.2% N, N'-methylene bisacrylamide, dissolved in 0.1 M sodium phosphate - 2.5 M urea buffer, pH 7.4, containing 0.1% SDS. Polymerization was accomplished by the addition of 0.3 ml of 10% ammonium persulfate (w/v) and 0.03 ml of N, N, N ~, N'-tetramethylethylenediamine to 40 ml of the acrytamide solution. Samples containing 250-300 gg protein were dissolved in 100 gl 5 M urea, 0.01 M sodium phosphate and applied to the gels. Electrophoresis was carried out at 50 volts, 5 ° C for 30 h (ttoefer Scientific Instruments, San Francisco, Calif., U.S.A.) The gels were stained in 0.2% (w/v) amido black in water-methanol-acetic acid (6:3:1, v/v/v) for 60 minutes. After destaining in water-methanol-acetic acid (6: 3:1, v/v/v) for two days, the gels were scanned with an Ortec Densitometer No. 4310. In some experiments, the cells had been labelled with L4C-leucine for studies on NHP turnover or with 3ZP-orthophosphate in order to study phosporylation of chromatin proteins [27].
Fig. 1. Comparative analyses of phenol-soluble chromatin proteins from various tissues of the rat. This experiment served as a control for comparative studies on phenol-soluble chromatin proteins from various human leukaemia cells and normal leukocytes. It is demonstrated that most proteins were common to all tissues studied and that tissue specificity of this protein fraction appears to be related to quantitative differences in the distribution profile. For technical details see Fig. 2
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s. Seeber et al. : Nonhistone Chromatin Proteins in Normal and LeukaemicHuman Leukocytes
tive distribution of these proteins. Compared to the studies of Teng et at. (1971), more individual proteins in the high molecular weight region were isolated using citric acid nuclei. Most of the proteins isolated were present in all four tissues studied, but the protein content per individual band varied largely from tissue to tissue. It was, however, not possible to demonstrate single proteins specific for liver, kidney, spleen or brain.
2. Chromatin Proteins in Normal Lymphocytes and Normal Granuloeytes Phenol-soluble chromatin proteins from normal granulocytes and normal lymphocytes are shown in Fig. 2. As the main difference, band 25 was present in granulocytes but not in lymphocytes in three individual experiments. Conversely, band 26a was found in lymphocytes but not in granulocytes. All other protein bands were distributed similarly in both cell types.
3. Chromatin Proteins in Leukaemic Lymphocytes When compared to normal lymphocytes, no additional bands were noted for CLL lymphocyte N H P patterns, but the concentration of some proteins in the high molecular weight area (bands 7-11) appeared to be increased in the leukaemic cells. 32p-labelling patterns of cells from CLL, A L L and leukaemic lymphosarcoma are presented in Fig. 3. Higher label in most of the bands was noted in the lymphosarcoma cells. These cells were also characterized by significantly higher concentrations in the individual bands (Figs. 3 and 4). The patterns also demonstrated that some of the phenol-soluble proteins are no phosphoproteins and that the amount of phosphorylation per mg protein varied widely from band to band.
4. Chromatin Proteins in Various Acute Leukaemia Blast Cells Phenol-soluble chromatin proteins from a variety of patients with acute lenkaemia are shown in Fig. 4. The patterns of amido black stained proteins demonstrate a remarkable similarity within the subgroups of acute human leukaemia as defined by morphology and cytochemical techniques. It may be of special interest that the chromatin proteins from C M L blast crisis, the terminal acute phase of chronic myeloid leukaemia, are highly similar when compared to all A M L patterns. A number of authors have argued that the blast crisis of C M L may be a special form of acute myeloid leukaemia. By cytogenetic analyses
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Fig. 2. Phenol-solublechromatin proteins extracted from citric acid nuclei (0.25% citric acid) of human normal lymphocytes (NL) and normal granulocytes(NG). Samplescontaining 200 ~tg protein were separated on 0.6 x 14 cm 10% polyacrylamidegels composed of 10% acrylamide,0.2% N, N'-methylenebisacrylamide, dissolved in 0.1 M sodium phosphate/2,5 M urea buffer, pH 7.4, containing 0.1% SDS. Electrophoresis was carried out at 50 volts, 5°C for 30 h (Hoefer Scientific Instruments, San Francisco, Calif., USA). The gels were stained with amido black and scanned with an Ortec Densitometer No. 4310
(Prof. Dr. Hossfeld, Essen), the C M L blast cells analysed here were derived from a patient with two typical Philadelphia chromosomes and a hyperdiploidy of 47-48 chromosomes in most metaphases. Apparently, if some proteins were attached to the chromatin of Philadelphia chromosomes, they could not be resolved with these techniques.
5. Differences of Chromatin Proteins from Myelocytes and Granulocytes A comparison of chromatin proteins from cells of different maturation stages derived from chronic my-
S. Seeber et al. : Nonhistone Chromatin Proteins in Normal and Leukaemic Human Leukocytes
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Fig. 3. Comparative analysis of phenol-soluble chromatin proteins from chronic lymphocytic le@aemia (CLL), acute lymphoblastic leukaemia (ALL) and leukaemic lymphosarcoma (LLS). Solid lines: densitometer tracings; dotted lines: distribution of 3zp radioactivity
Fig. 4. Comparative electrophoretic banding patterns of phenol-soluble chromatin proteins from various human acute leukaemia cells and cells of chronic myeloid leukaemia blast crisis. The bands were labelled with increasing electrophoretic mobility
Fig. 5A and B. Comparative analysis of phenol-soluble chromatin proteins from human chronic myeloid leukaemia myelocytes (A) and leukaemic granulocytes isolated from the same patient (B). 1.5 x 109 cells both of myelocytes and granulocytes were separately incubated in a phosphate-free medium (Seeber and Schmidt, 1973) together with 32p-orthophosphate (2.5 mCi, 50 ml cell suspension, 1 h, 37 ° C). Following incubation, citric acid nuclei were prepared (Taylor et al., 1973) and chromatin proteins were isolated according to the method of Teng et al. (1971). The details of the electrophoretic separation are presented in Fig. 2. For determination of 32p radioactivity the gels were frozen at - 7 0 ° C and cut into 1 mm slices
Fig. 6. Specific interaction of adriamycin (A: control; B: 10 gg/ml A D M ; C: 20 gg/ml A D M ; D: 40 gg/ml ADM) with phenol-soluble chromatin proteins of human acute myelomonocytic leukaemic cells. The cells were incubated with adriamycin in a phosphate-free medium for 2 h at 37 ° C. At the end of this incubation the cells were washed twice in N K M buffer, resuspended in a phosphate-free medium and incubated together with 50 gCi/ml 32p-orthophosphate for 4 h at 37 ° C. Phenol-soluble chromatin proteins were extracted from citric acid nuclei and separated as described in Fig. 2
s. Seeber et al. : NonhistoneChromatinProteins in Normal and LeukaemicHuman Leukocytes eloid leukaemia is presented in Fig. 5. Virtually all chromosomal proteins present in myelocytes and promyelocytes were also present in mature (leukaemic) granulocytes. However, as a remarkable feature already found in nonleukaemic normal granulocytes, one nonhistone protein (band no. 25) not found in the proliferating and differentiating myelocytes was present in preparations from mature leukaemic granulocytes. As a second result of this experiment, phosphorylation of phenol-soluble chromatin proteins in myelocytic cells appears to be inversely related to the maturation stage.
6. Selective Changes of Chromatin Proteins' by Intercalating Agents (Adriamycin and Daunomycin) Fig. 6 shows the densitometer tracings of phenol-soluble chromatin proteins and the distribution of 14Cleucine label after incubation of myetomonocytic leukaemia cells with increasing adriamycin concentrations for 2 hours. The protein designated as no. 30 was selectively decreased in a dose-dependent fashion. This protein labelled well with 14C-leucine but, following 3~p-labelling data, is apparently not phosphorylated. In a comparative study no effects on NHP were noted following exposure of leukaemia cells to bleomycin (30 ~tg/ml) or actinomycin D (2.5 gg/ml).
Discussion
The extraction, separation and electrophoretic analysis of phenol-soluble acidic proteins associated with nuclear DNA has first been described by Teng et al. (1971). Most of these proteins have been shown to be phosphoproteins which incorporate 3Zp-orthophosphate in vivo leading to formation of phosphothreonine and phosphoserine residues. Teng et al. (1971) could demonstrate that a) the patterns of phosphorylation of individual proteins varied from tissue to tissue, b) binding of these proteins with DNA was species-specific, and c) some of these phosphoproteins stimulated transcription in a cell-free RNA-synthesizing system. Originally, this class of nuclear proteins was isolated from 0.32 M sucrose-3mM MgClz nuclei [36]. In our own studies on phenol-soluble proteins from various human leukaemia and lymphoma cells, a citric acid procedure was used since MacGillivray et al. (t972) had demonstrated that "sucrose nuclei" contained fewer high molecular weight species compared to "citric acid nuclei" in most tissues studied. Comparative analyses of phenol-soluble chromatin proteins in human leukocytes have not been reported in the literature. Very recently, two-dimensional gel electrophoresis studies of "Chromatin frac-
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tion II" proteins - a different protein fraction including more than 90% of the total chromatin-associated nonhistone proteins - were presented for normal lymphocytes, PHA-stimulated Iymphocytes and teukaemic lymphoblasts [44]. In these studies, some high molecular weight chromatin proteins were found increased in stimulated compared to normal lymphocytes. In addition, leukaemic lymphoblasts (ALL) were characterized by lower concentrations of individual fast moving proteins (A24, BA) and by enhanced protein concentrations in distinct spots of the high molecular weight region. However, a patient-to-patient variation was noted in these ALL patients which had been earlier observed in studies on acidic nuclear proteins from CML, CLL and AML cells (Weisenthal and Ruddon, 1972; 1973) probably related to protease activity. Nevertheless, these authors had demonstrated characteristic protein banding patterns in a comparison of different human leukaemic diseases [39]. It should be noted in this context that differences of chromatin proteins may be introduced by a different distribution of cells in the various stages of the cell cycle. Stein et al. (1975) have shown that HeLa S-phase chromatin contained a nonhistone protein which could render the histone genes available for transcription. This protein was not found in Gl-phase HeLa cells [33]. NHP changes as a function of various stages of the cell cycle have also been reported by other authors [20, 32]. Several conclusions may be drawn from the data presented in Figs. 1 to 6. It is apparent that the protein fraction analysed has some tissue specificity. The characteristic differences between normal lymphocytes and normal granulocytes with at least one specific protein (no. 25 in granulocytes, no. 26a in lymphocytes) have initiated further studies on the chemistry and function of these components. Protein no. 25 appears to be of special interest since it has been found in normal and leukaemic granulocytes but not in myelocytes (Fig. 5) or in lymphocytes. As both myelocytes and lymphocytes, under certain conditions, are able to divide, this fraction might represent some gene control protein (Fig. 5). The high number and concentration of high molecular weight proteins in the lymphosarcoma cells may reflect to some extent the high-grade neoplastic nature of these cells. In earlier comparisons of liver and hepatoma cells, a similar increase of high molecular nonhistone proteins has been described for highly malignant tumour cells [42, 43]. The labelling data from the experiment illustrated in Fig. 3 further suggest a relationship between the almost uniformly distributed phosphorylation of these phenol-soluble proteins and mitotic activities. The dose-dependent selective decrease of individual chromatin proteins after incubation with an inter-
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s. Seeber et at. : Nonhistone Chromatin Proteins in Normal and Leukaemic Human Leukocytes
c a l a t i n g a g e n t is a f i n d i n g o f special i n t e r e s t (Fig. 6). T h i s effect h a s also b e e n n o t i c e d in N o v i k o f f h e p a t o m a cells [30]. T h e d a t a i n d i c a t e t h a t a d r i a m y c i n a n d d a u n o m y c i n m a y exert a specific d a m a g e o n the n u c l e a r c h r o m a t i n o f v a r i o u s t u m o u r cells, d i f f e r e n t f r o m t h a t c a u s e d b y a c t i n o m y c i n D or b l e o m y c i n . T h e s t r u c t u r e o f the d a u n o m y c i n - D N A c o m p l e x h a s b e e n a n o b j e c t o f n u m e r o u s i n v e s t i g a t i o n s . O n the a s s u m p t i o n t h a t the m o d e l o f P i g r a m et at. (1972) is c o r r e c t - r e c e n t l y s u p p o r t e d b y D i M a r c o a n d A r c a m o n e (1975) - it c o u l d be c o n c l u d e d t h a t p r o t e i n no. 30, w h i c h was affected b y d a u n o m y c i n b o t h in the e x p e r i m e n t a l t u m o u r a n d the h u m a n l e u k a e m i a cells, s h o u l d be a m a r k e r p r o t e i n for t h e d a u n o m y c i n i n t e r c a l a t i o n site at the m a j o r g r o o v e of the D N A d o u b l e - h e l i x . Its d o s e - d e p e n d e n t decrease in p a t t e r n s o f tightly b o u n d , p h e n o l - s o l u b l e p r o t e i n s c o u l d be explained by a loosening of some DNA-protein bonds f o l l o w i n g d r u g i n t e r c a l a t i o n a n d local d i s t o r t i o n . T h u s , p r o t e i n no. 30 w o u l d be e l u t e d f r o m the c h r o m a t i n a l r e a d y at early steps o f the e x t r a c t i o n p r o c e dure. A n o t h e r e x p l a n a t i o n c o u l d be t h a t d u e to crosslinks o f d o u b l e - s t r a n d e d D N A a t the i n t e r c a l a t i o n site a n d d u e to a n a l t e r e d t e r t i a r y s t r u c t u r e o f the DNA, some proteins could become trapped. Both mechanisms would prevent individual proteins from e n t e r i n g t h e p h e n o l p h a s e f r o m w h i c h t h e final p r o t e i n f r a c t i o n was i s o l a t e d by dialysis.
References
1, Arnold, E.A., Buksas, M.M., Young, K.E.: A comparative study of some properties of chromatin from two "minimal deviation" hepatomas. Cancer Res. 33, 1169-1176 (1973) 2. Barrett, T., Maryanka, D., Hamlyn, P.H., Gould, H.J. : Nonhistone proteins control gene expression in reconstituted chromatin. Proc. nat. Acad. Sci. USA 71, 5057-5061 (1974) 3. Biessmann, H., Rajewsk?¢, M.F.: Nuclear protein patterns in developing and adult brain and in ethylnitrosourea-induced neuroectodermal tumours of the rat. J. Neurochem. 24, 387-393 (1975) 4. Boyum, A. : Separation of leucocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, Suppl. No. 97 (1968) 5. Busch, H., Ballal, N.R., Olson, M.O.J., Yeoman, L.C.: Chromatin and its nonhistone proteins. In: Methods in Cancer Research (H. Busch, ed.), Vol. 11, pp. 43-121. New York: Academic Press 1975 6. Chae, C.B., Smith, M.C, Morris, H.P. : Chromosomal nonhistone proteins of rat hepatomas and normal rat liver. Biochem. biophys. Res. Commun. 60, 1468-1474 (1974) 7. Chiu, J.F., Tsai, Y.H., Sakuma, K., Hnilica, L.S. : Regulation of in vitro mRNA transcription by a fraction of chromosomal proteins. J. bioI. Chem. 250, 9431-9433 (1975) 8. Desai, L.S. : Mechanism of gene interaction and histone deacetylation in human leukemic cells. J. Cell Biol. 63, 82a, Abstr. no. 163 (1974) 9. Desai, L.S., Wulff, U.C., Foley, G.E. : Properties of chromosomal proteins of human leukemic cells. Biochimie 57, 315-324 (1975)
10. Di Marco, A., Arcamone, F.: DNA complexing antibiotics - daunomycin, adriamycin and their derivatives. Arzneim.Forsch. 25, 368-375 (1975) 11. Dounce, A.L. : The isolation and composition of cell nuclei and nucleoli. In: The Nucleic Acids. (E. Chargaff and J.N. Davidson, eds.), Vol. 2, pp. 93 153. New York: Academic Press 1955 12. Johnson, E.M., Karn, J., Allfrey, V.G. : Early nuclear events in the induction of lymphocyte proliferation by mitogens. Effects of concanavalin A on the phosphorylation and distribution of nonhistone chromatin proteins. J. biol. Chem. 249, 4990-4999 (1974) 13. Kostraba, N.C., Wang, T.Y.: Differential activation of transcription of chromatin by non-histone fractions. Biochim. biophys. Acta (Amst.) 262, 169-180 (1972) 14. Levy, R., Levy, S., Rosenberg, S.A., Simpson, R.T.: Selective stimulation of nonhistone chromatin protein synthesis in lymphoid cells by phytohemagglutinin. Biochemistry 12, 224-228 (1973) 15. MacGitlivray, A.J., Carroll, D., Paul, J.: The heterogeneity of the non-histone chromatin proteins from mouse tissues. FEBS Lett. 13, 204-208 (197I) 16. MacGillivray, A.J, Cameron, A., Krauze, R.J., Rickwood, D., Paul, J.: The non-histone proteins of chromatin. Their isolation and composition in a number of tissues. Biochim. biophys. Acta (Amst.) 277, 384.402 (1972) 17. Marushige, K., Bonnet, J. : Template properties of liver chromatin. J. molec. Biol. 15, 160-174 (1966) 18. Marushige, K., Brutlag, D., Bonner, J. : Properties of chromosomal nonhistone protein of rat liver. Biochemistry 7, 314%3155 (1968) 19. Masera, P., Pileri, A., Brachet, J., Hulin, N.: Lymphocyte actinomycin binding capacity in chronic lymphocytic leukaemia. Experientia (Basel) 28, 1484 (1972) 20. McClure, M.E., Hnilica, L.S. : Nuclear proteins in genetic restriction. III. The cell cycle. Sub-cell. Biochem. 1, 311 332 (1972) 21 Nood6n, L.D:, van den Broek, H.W.J., Sevall, J.S. : Stabilization of histones from rat liver. FEBS Lett. 29, 326-328 (1973) 22. Paul, J., Gilmour, R.S. : Organ-specific restriction of transcription in mammalianchromatin. J. molec. Biol, 34, 305-316 (1968) 23. Pigram, W.J., Fuller, W., Hamilton, L.D.: Sterochemistry of intercalation: interaction of daunomycin with DNA. Nature, New Biol. 235, 1%19 (1972) 24. Pileri, A., Masera, P., Hulin, N. : Brachet, J.: Actinomycin binding capacity in human leukaemic lymphoid cells. Acta haemat. (Basel) 48, 89-97 (1972) _ 2 5 . Platz, R.D., Kish, V.M., Kleinsmith, L.J.: Tissue specificity of non-historic chromatin phosphoproteins. FEBS Lett. 12, 38~40 (1970) 26. Sawada, H., Gilmore, V.H., Saunders, G.F.: Transcription from chromatins of human lymphocytic leukemia cells and normal lymphocytes. Cancer Res. 33, 428-434 (1973) 27. Seeber, S., Schmidt, C,G. : Isolation of rapidly labelled nuclear RNA of high specific activity from human leukaemic cells. Klin. Wschr. 51, 677-679 (1973) 28. Seeber, S,, Brucksch, K.P., K~iding, J., Schmidt, C.G., Busch, H.: Oligonucleotides of ribosomal 28 S RNA in human leukemic cells and normal lymphocytes. Cancer Res. 34, 1281-1288 (t974a) 29. Seeber, S., Kfiding, J., Brucksch, K.P., Schmidt, C.G.: Defective rRNA synthesis in human leukaemic blast cells? Nature (Loud.) 248, 673-675 (1974b) 30. Seeber, S., Schmidt, C.G., Busch, H.: Isolation, separation and fractionation of human leukemic and normal teukocytes. Comparative studies on preribosomal and ribosomal RNA and on non-histone chromatin proteins. In: Methods in Cancer Research (H. Busch, ed.), Vol. 14, pp. 131 t90. NewYork: Academic Press t978
S. Seeber et al. : Nonhistone Chromatin Proteins in Normal and Leukaemic Human Leukocytes 3 i. Spelsberg, T.C, Hnilica, L.S., Ansevin, A.T. : Proteins of chromatin in template restriction. III. The macromolecules in specific restriction of the chromatin DNA. Biochim. biophys. Acta (Amst.) 228, 550-562 (197l) 32. Stein, G.S., Borun, T.W. : The synthesis of acidic chromosomal proteins during the celI cycle of HeLa S-3 cells. I. The accelerated accumulation of acidic residual nuclear protein before the initiation of DNA replication. J. Cell Biol. 52, 292-307 (1972) 33. Stein, G.S., Park, W., Thrall, C., Mans, R., Stein, J.: Regulation of cell cycle stage-specific transcription of histone genes from chromatin by non-histone chromosomal proteins. Nature (Lond.) 257, 764-767 (1975) 34. Taylor, C.W., Yeoman, L.C, Daskal, I., Busch, H.: TwodimensionaI electrophoresis of proteins of citric acid nuclei prepared with aid of a Tissumizer®. Exp. Cell Res. 82, 2t 5-226 (t973) 35. Teng, C.S., Hamilton, T.H.: Role of chromatin in estrogen action in the uterus. II. Hormone-induced synthesis of nonhistone acidic proteins which restore histone-inhibited DNA-dependent RNA synthesis. Proc. nat. Acad. Sci. USA 63, 465-472 (1969) 36. Teng, C.S., Teng, C.T., Allfrey, V.G. : Studies of nuclear acidic proteins. Evidence for their phosphorylation, tissue specificity, selective binding to deoxyribonucleic acid, and stimulatory effects on transcription. J. biot. Chem. 246, 3597 3609 (t97l) 37. Vifiuela, E., Algranati, I.D., Ochoa, S.: Synthesis of virusspecific proteins in Escherichia coli infected with the RNA bacteriophage MS2. Europ. J. Biochem. 1, 3-11 (1967) 38. Wang, T.Y. : Restoration of histone-inhibited DNA-dependent RNA synthesis by acidic chromatin proteins. Exp. Cell Res. 53, 288-291 (1968) 39. Weisenthal, L.M., Ruddon, R,W.: Characterization of human
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leukemia and Bnrkitt lymphoma cells by their acidic nuclear protein profiles. Cancer Res. 32, 1009 1017 (1972) 40. Weisenthal, L.M., Ruddon, R.W.: Catabolism of nuclear proteins in control and phytohemagglutinin-stimulated human lymphocytes, leukemic leukocytes, and Burkitt lymphoma cells. Cancer Res. 33, 2923-2935 (1973) 41. Wu, F.C., Elgin, S.C.R., Hood, L.E.: Nonhistone chromosomal proteins of rat tissues. A comparative study by gel electrophoresis. Biochemistry 12, 2792-2797 (1973) 42. Yeoman, L,C., Taylor, C.W., Jordan, J.J., Busch, H.: Twodimensional polyacrylamide gel electrophoresis of chromatin proteins of normal rat liver and Novikoff hepatoma ascites cells. Biochem. biophys. Res. Commun. 53, t067-1076 (1973) 43. Yeoman, L,C., Taylor, C.W., Jordan, J.J., Busch, H.: Differences in chromatin proteins of growing and non-growing tissues. Exp. Cell Res. 91, 207-215 (1975) 44. Yeoman, L.C., Seeber, S., Taylor, C.W., Fernbach, D.J., FaIletta, J.M., Jordan, J.J., Busch, It.: Differences in chromatin proteins of resting and growing human Iymphocytes. Exp. Ceil Res. 100, 47-55 (I976)
Received September 15, 1977 Accepted October 5, 1978 Priv.-Doz. Dr. S. Seeber Innere Universit~itsklinik und Poliklinik (Tumorforschung) Westdeutsches Tumorzentrum Hufelandstrage 55 D-4300 Essen Federal Republic of Germany
Klin. Wochenschr. 57, 257-265 (1979)
'9 Springer-Verlag 1979
Originalien Comparative Studies on Phenol-Soluble Nonhistone Chromatin Proteins in Normal and Leukaemic Human Leukocytes* S. Seeber, T. Meshkov**, K.P. Brucksch, J. K/iding, C.G. Schmidt and H. Busch Inhere Universitfitsklinik und Poliklinik (Tumorforschung), Westdeutsches Tumorzentrum, Essen Department of Pharmacology, Baylor College of Medicine, Houston, TX, USA
Vergleichende Untersuehungen fiber phenoMOsliehe Chromatinproteine in normalen und leuk~imischen menschlichen Leukozyten Zusammenfassung. Phenol-16sliche Chromatinproteine wurden aus Zellkernen normaler und leukfimischer menschlicher Leukozyten isoliert. Die Proteine, deren Bedeutung im Rahmen der Kontrolle des Transkriptionsvorganges diskutiert wird, wurden durch Analyse ihrer geMektrophoretischen Verteilungsmuster und ihrer Markierung mit 14C-Leuzin oder 32p-Orthophosphat verglichen. AuBerdem wurde der Einflul3 interkalierender Agentien wie Adriamycin untersucht. Die Fraktion phenol-16slicher Nicht-HistonProteine des Chromatins menschlicher Leukozyten enth/ilt mindestens 20-35 individuelle Proteine, welche mit zunehmender Laufgeschwindigkeit auf 10%Polyacrylamidgelen numeriert wurden. Zwischen normalen Lymphozyten und normalen Granulozyten ergaben sich charakteristische Unterschiede mit jeweils einer spezifischen Proteinbande (Nr. 26a bei Lymphozyten; Nr. 25 bei Granulozyten) ftir beide Zelltypen. Interessanterweise war das Protein Nr. 25 in Myelozyten (CML) nicht vorhanden, jedoch in reifen Granulozyten desselben Patienten (CML) nachweisbar. Die radioaktiven Markierungen der Proteine mit 32p (Phosphorilierung) bzw. 14C-Leuzin (Umsatz) nahmen ganz allgemein mit zunehmender Zelldifferenzierung ab. Leuk/imische Lymphozyten unterschieden sich yon normalen Lymphozyten durch erh6hte Protein-Konzentrationen im hochmolekularen Bereich. Beim Vergleich von Zellen aus AML, ALL * These studies were supported by grant Se 161/6 from Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg ** Supported by Humboldt-Stiftung Offprint requests to: Priv.-Doz. Dr. S. Seeber (address see p. 265)
und der CML-Blastenkrise ergaben sich bei weitgehend fihnlichen Mustern keine typischen Differenzen. Ebenso waren gemeinsame leukfimiespezifische Abweichungen gegenfiber Normalzellen nicht nachzuweisen. Lymphosarkomzellen zeigten allerdings quantitative und qualitative Abweichungen vom gew6hnlichen Verteilungsmuster der Zellkernproteine. Nach Einwirkung von Adriamycin in vitro kam es bei den verschiedensten Zellinien dosisabhfingig zu einer selektiven Abnahme eines bestimmten Proteins (Nr. 30) dessen m6gliche Funktion als ,,marker" fiir den Interkatationslocus von Adriamycin an der DNA diskutiert wird.
Sehliisseiwiirter: Phenol-16sliche Chromatinproteinemenschliche Leukfimiezellen - Adriamycin. Summary. Phenol-soluble chromatin proteins which may be involved in gene control mechanisms have been isolated from citric acid nuclei of normal and leukaemic human leukocytes derived from freshly obtained venous blood. They were compared by onedimensional gel electrophoresis and by comparative labelling with ~4C-leucine or 32p-orthophosphate. In addition, the influence of intercalating agents such as adriamycin and daunomycin was studied. Between 20-35 individual proteins were found in the phenol-soluble nonhistone protein fraction of human leukocytes. They were numbered with increasing mobility on 10% polyacrylamide gels. Distinct differences were found between normal lymphocytes and normal granulocytes, with one specific protein (no. 26a in lymphocytes; no. 25 in granulocytes) for both cell types. Interestingly, protein no. 25 was not present in CML myelocytes but in CML granulocytes. 32p_ and leucine labels were generally found decreased
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S. Seeber et al. : Nonhistone Chromatin Proteins in Normal and LeukaemicHuman Leukocytes
with increasing cell differentiation. Leukaemic lymphocytes differed from normal lymphocytes by increased protein concentrations in the high molecular weight region. In comparisons of A M L and ALL cells, including blast cells of C M L blast crisis, no constant differences nor any markers common to leukaemic cells as compared to normal cells were detectable. However, lymphosarcoma cells showed quantitiative and qualitative aberrations from the usual leukaemia pattern. After in vitro incubations of cells with adriamycin a selective decrease of an individual protein (no. 30) was noted. This protein may serve as a marker for the intercalation site.
Key words: Phenol-soluble chromatin proteins - Human leukaemia cells - Adriamycin.
Only few definitive studies have been reported on nonhistone chromatin proteins in human leukocytes [5, 8, 9, 12, 14, 39, 40, 44]. Analyses of nuclear acidic proteins in various human normal and leukaemic cell lines gain importance since it has been shown that some of these proteins are specific in different tissues and in different species [15, 25, 38, 41, 42]. It has been derived from transcriptional studies that nonhistone proteins are probably involved in positive control mechanisms for gene expression [2, 7, 13, 18, 22, 31, 35]. More recently, it has been suggested that differences exist in nonhistone proteins between chromatin of normal cells and tumour cells that may be related to altered gene expression [1, 3, 5, 6, 44]. Earlier studies have shown that chromatin from human leukaemic lymphocytes differed from that of normal lymphocytes by a decreased actinomycin D binding capacity [19, 24] and by a higher template activity [26]. In addition, histone deacetylase activity has been shown to be greater in lymphocytes from leukaemic patients, and the protein responsible for this activity was shown to bind intimately to isolated chromatin [8]. Very recently, biophysical and biochemical characteristics of isolated chromatin and individual chromosomal components of cultured human lymphocytes from normal donors and from patients with infectious mononucleosis and acute lymphoblastic leukaemia were presented [9]. Lymphocyte chromatin had been isolated following the method of Marushige and Bonnet (1966), using homogenization of cells in saline-EDTA buffer and in 0.01 M Tris buffer (pH 8.0) followed by purification of chromatin by centrifugation through 1.7 M sucrose (0.01 M Tris, pH 8.0). These studies showed that a) after thermal
denaturation higher Tm values were found in the leukaemic chromatin, b) differences were present between leukaemic and normal lymphocytes in the ratios o f D N A : RNA: protein, and c) the ratio of nonhistone proteins to histone proteins associated with chromatin was higher in the leukaemic cell lines [9]. In this comparison, amino-acid compositions of the histones were very similar, but the nonhistone chromosomal proteins appeared to be more heterogeneous with respect to amino-acid composition. Also, onedimensional densitometer tracings had revealed considerable heterogeneity when different kinds of human lymphocytic cells were compared [9]. Chromosomal acidic proteins isolated from the purified chromatin of human leukaemic cells greatly stimulated the template activity of the chromatin in in vitro synthesis [9]. Our own studies were begun in order to compare nonhistone chromatin protein patterns from various human leukaemia cells and normal teukocytes which were in all cases freshly obtained from untreated patients. This seemed to be important since after longterm culture the leukaemic nature of cultured leukocytes may be questionable. The goal was to define possible chromatin markers for individual leukaemic cell lines and to search for specific differences between myelocytic and lymphocytic cells which are, at the blast level, often not distinguishable by morphological criteria.
Materials and Methods 1. Separation of Leukocytes fmm Erythrocytes
Freshly drawn heparinized blood was diluted 1:1 (v/v) with neoPlasmagel (3% gelatin, tool. wt 35,000, Na + 142mval/L, Ca ++ 2.8 mval/L, C1- 109mval/L; B. Braun AG, Melsungen, F.R.G.). The mixture was allowed to settle in a siliconized glass cylinder at 37° C for 30-40 min. After sedimentation, both the supernatant and the buffy coat were carefully removed with a Pasteur pipette. The cells were centrifuged for 10 min at 1,000rpm, RC3 Sorvall at 4° C and washed 2 to 3 times with NKM buffer (0.13 M NaC1, 0.005 M KC1, 0.008 M Mg C12). 2. Separation of Mononuclear Cellsfrom Granulocytes
In a one-stage procedure as originally designed by Boyum (1968), separations were carried out with a sodium metrizoate-Ficollmixture (Lymphoprep,density 1.077_+0.001g/ml, 9.6% (w/v) sodium metrizoate, 5.6% (w/v) Ficoll; Pharmacia, Uppsala, Sweden). 3. Isolation of Leukocyte Nuclei with Intact Macromolecutes
A modified citric acid procedure similar to that first described by Dounce (1955) was employed. In studies on nuclear proteins it had been demonstrated that some chromatin proteins were re-
S. Seeber et al. : Nonhistone Chromatin Proteins in Normal and Leukaemic tluman Leukocytes leased from the nucleus when citric acid acid concentrations between 1.5 and 5% were used [34]. These studies had shown that with a dilution of 0.025 M citric acid no visible alterations of the nuclear morphoIogy were detectable and only few, if any, chromatin nonhistone proteins were released selectively, as assayed by the 0.4 N sulfuric acid extract. In addition, the chances for proteolytic degradation during the procedure of nuclear isolation were reduced at the low pH (2.5) maintained during all steps of preparation [44]. Other studies using human leukaemia cells had pointed out the serious degradation problem [40]. NKM-buffer washed leukocytes were suspended in 10 volumes (w/v) of 0.025 M citric acid (pH 2.5) and disrupted with a Super Dispax Model SDT-182 in twelve (lymphocytic cells, myeloblasts) to eighteen (myelocytes, granulocytes) 15s bursts (3 to 4~/2 min of disruption) at a Regler setting of 7 [16, 34]. Crude nuclei were purified through 0.88 M sucrose containing 3.3 mM Ca 2+ and 100 gM PMSF (Sigma Chemical Corporation, St. Louis, Mo., U.S.A.). The nuclear pellet was washed in a buffer containing 0.15 m NaCI, 100 gM PMSF and 0.01 M Tris, pH 7.2 and frozen (Nood6n et al., 1973).
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Results 1. Tissue-SpecS'city o f Phenol-Soluble Chromatin Proteins F r o m e a r l i e r s t u d i e s o f T e n g e t al. ( 1 9 7 1 ) c o m p a r i n g p h e n o l - s o l u b l e c h r o m a t i n p r o t e i n s i n r a t liver, k i d n e y , s p l e e n a n d b r a i n , it h a d b e e n c o n c l u d e d t h a t m a n y of the proteins isolated were tissue-specific. In a control experiment for studies in human leukocytes, citric a c i d n u c l e i w e r e p r e p a r e d f r o m r a t liver, k i d n e y , spleen and brain, and phenol-soluble chromatin p r o t e i n s w e r e i s o l a t e d . A s d e m o n s t r a t e d i n Fig. 1, characteristic differences were noted in the quantita-
4. Isolation of Phenol-Soluble Chromatin Proteins .from Human Leukocytes Purified nuclear pellets from leukaemia cells and normal leukocytes were extracted two times with 0.14 M NaC1. Following centrifugation for 5 min at 3,000 g for 5 rain, the extraction was repeated. The nuclear residue was suspended in 10 volumes of 1:1 (v/v) chloroform-methanol containing 0.2 N HC1. After centrifugation at 5,000 g for 5 min, the sediment was extracted with 10 volumes of 2:1 chloroform-methanol containing 0.2 N HC1 and centrifuged as before. The pellet was washed with ether, resuspended in 5 volumes of a buffer containing 0.1 M Tris-HC1, 0.01 M EDTA and 0.14 M 2-mercaptoethanot, pH 8.4. An equal volume of buffersaturated cold phenol was added and the suspension was allowed to stand for 14 h at 2 ° C. After centrifugation of this mixture (10 rain, 12,000 g), the aqueous phase was re-extracted with an equal volume of phenol and centrifuged as before. The combined phenol phases were dialysed in a three-step procedure that followed exactly the conditions described by Vifiuela et al. (1967) and Teng et al. (197I). The phenol-soluble proteins were thus restored to the aqueous phase and ready for further characterization.
5. Separation of Phenol-Soluble Chromatin Proteins by Analytical One-Dimensional Gel Electrophoresis Partial resolution of the complex mixture of phenol-soluble chromatin proteins was achieved on 0.6 x 14 cm analytical gels composed of 10% acrylamide, 0.2% N, N'-methylene bisacrylamide, dissolved in 0.1 M sodium phosphate - 2.5 M urea buffer, pH 7.4, containing 0.1% SDS. Polymerization was accomplished by the addition of 0.3 ml of 10% ammonium persulfate (w/v) and 0.03 ml of N, N, N ~, N'-tetramethylethylenediamine to 40 ml of the acrytamide solution. Samples containing 250-300 gg protein were dissolved in 100 gl 5 M urea, 0.01 M sodium phosphate and applied to the gels. Electrophoresis was carried out at 50 volts, 5 ° C for 30 h (ttoefer Scientific Instruments, San Francisco, Calif., U.S.A.) The gels were stained in 0.2% (w/v) amido black in water-methanol-acetic acid (6:3:1, v/v/v) for 60 minutes. After destaining in water-methanol-acetic acid (6: 3:1, v/v/v) for two days, the gels were scanned with an Ortec Densitometer No. 4310. In some experiments, the cells had been labelled with L4C-leucine for studies on NHP turnover or with 3ZP-orthophosphate in order to study phosporylation of chromatin proteins [27].
Fig. 1. Comparative analyses of phenol-soluble chromatin proteins from various tissues of the rat. This experiment served as a control for comparative studies on phenol-soluble chromatin proteins from various human leukaemia cells and normal leukocytes. It is demonstrated that most proteins were common to all tissues studied and that tissue specificity of this protein fraction appears to be related to quantitative differences in the distribution profile. For technical details see Fig. 2
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s. Seeber et al. : Nonhistone Chromatin Proteins in Normal and LeukaemicHuman Leukocytes
tive distribution of these proteins. Compared to the studies of Teng et at. (1971), more individual proteins in the high molecular weight region were isolated using citric acid nuclei. Most of the proteins isolated were present in all four tissues studied, but the protein content per individual band varied largely from tissue to tissue. It was, however, not possible to demonstrate single proteins specific for liver, kidney, spleen or brain.
2. Chromatin Proteins in Normal Lymphocytes and Normal Granuloeytes Phenol-soluble chromatin proteins from normal granulocytes and normal lymphocytes are shown in Fig. 2. As the main difference, band 25 was present in granulocytes but not in lymphocytes in three individual experiments. Conversely, band 26a was found in lymphocytes but not in granulocytes. All other protein bands were distributed similarly in both cell types.
3. Chromatin Proteins in Leukaemic Lymphocytes When compared to normal lymphocytes, no additional bands were noted for CLL lymphocyte N H P patterns, but the concentration of some proteins in the high molecular weight area (bands 7-11) appeared to be increased in the leukaemic cells. 32p-labelling patterns of cells from CLL, A L L and leukaemic lymphosarcoma are presented in Fig. 3. Higher label in most of the bands was noted in the lymphosarcoma cells. These cells were also characterized by significantly higher concentrations in the individual bands (Figs. 3 and 4). The patterns also demonstrated that some of the phenol-soluble proteins are no phosphoproteins and that the amount of phosphorylation per mg protein varied widely from band to band.
4. Chromatin Proteins in Various Acute Leukaemia Blast Cells Phenol-soluble chromatin proteins from a variety of patients with acute lenkaemia are shown in Fig. 4. The patterns of amido black stained proteins demonstrate a remarkable similarity within the subgroups of acute human leukaemia as defined by morphology and cytochemical techniques. It may be of special interest that the chromatin proteins from C M L blast crisis, the terminal acute phase of chronic myeloid leukaemia, are highly similar when compared to all A M L patterns. A number of authors have argued that the blast crisis of C M L may be a special form of acute myeloid leukaemia. By cytogenetic analyses
Nk
N6
Fig. 2. Phenol-solublechromatin proteins extracted from citric acid nuclei (0.25% citric acid) of human normal lymphocytes (NL) and normal granulocytes(NG). Samplescontaining 200 ~tg protein were separated on 0.6 x 14 cm 10% polyacrylamidegels composed of 10% acrylamide,0.2% N, N'-methylenebisacrylamide, dissolved in 0.1 M sodium phosphate/2,5 M urea buffer, pH 7.4, containing 0.1% SDS. Electrophoresis was carried out at 50 volts, 5°C for 30 h (Hoefer Scientific Instruments, San Francisco, Calif., USA). The gels were stained with amido black and scanned with an Ortec Densitometer No. 4310
(Prof. Dr. Hossfeld, Essen), the C M L blast cells analysed here were derived from a patient with two typical Philadelphia chromosomes and a hyperdiploidy of 47-48 chromosomes in most metaphases. Apparently, if some proteins were attached to the chromatin of Philadelphia chromosomes, they could not be resolved with these techniques.
5. Differences of Chromatin Proteins from Myelocytes and Granulocytes A comparison of chromatin proteins from cells of different maturation stages derived from chronic my-
S. Seeber et al. : Nonhistone Chromatin Proteins in Normal and Leukaemic Human Leukocytes
261
Fig. 3. Comparative analysis of phenol-soluble chromatin proteins from chronic lymphocytic le@aemia (CLL), acute lymphoblastic leukaemia (ALL) and leukaemic lymphosarcoma (LLS). Solid lines: densitometer tracings; dotted lines: distribution of 3zp radioactivity
Fig. 4. Comparative electrophoretic banding patterns of phenol-soluble chromatin proteins from various human acute leukaemia cells and cells of chronic myeloid leukaemia blast crisis. The bands were labelled with increasing electrophoretic mobility
Fig. 5A and B. Comparative analysis of phenol-soluble chromatin proteins from human chronic myeloid leukaemia myelocytes (A) and leukaemic granulocytes isolated from the same patient (B). 1.5 x 109 cells both of myelocytes and granulocytes were separately incubated in a phosphate-free medium (Seeber and Schmidt, 1973) together with 32p-orthophosphate (2.5 mCi, 50 ml cell suspension, 1 h, 37 ° C). Following incubation, citric acid nuclei were prepared (Taylor et al., 1973) and chromatin proteins were isolated according to the method of Teng et al. (1971). The details of the electrophoretic separation are presented in Fig. 2. For determination of 32p radioactivity the gels were frozen at - 7 0 ° C and cut into 1 mm slices
Fig. 6. Specific interaction of adriamycin (A: control; B: 10 gg/ml A D M ; C: 20 gg/ml A D M ; D: 40 gg/ml ADM) with phenol-soluble chromatin proteins of human acute myelomonocytic leukaemic cells. The cells were incubated with adriamycin in a phosphate-free medium for 2 h at 37 ° C. At the end of this incubation the cells were washed twice in N K M buffer, resuspended in a phosphate-free medium and incubated together with 50 gCi/ml 32p-orthophosphate for 4 h at 37 ° C. Phenol-soluble chromatin proteins were extracted from citric acid nuclei and separated as described in Fig. 2
s. Seeber et al. : NonhistoneChromatinProteins in Normal and LeukaemicHuman Leukocytes eloid leukaemia is presented in Fig. 5. Virtually all chromosomal proteins present in myelocytes and promyelocytes were also present in mature (leukaemic) granulocytes. However, as a remarkable feature already found in nonleukaemic normal granulocytes, one nonhistone protein (band no. 25) not found in the proliferating and differentiating myelocytes was present in preparations from mature leukaemic granulocytes. As a second result of this experiment, phosphorylation of phenol-soluble chromatin proteins in myelocytic cells appears to be inversely related to the maturation stage.
6. Selective Changes of Chromatin Proteins' by Intercalating Agents (Adriamycin and Daunomycin) Fig. 6 shows the densitometer tracings of phenol-soluble chromatin proteins and the distribution of 14Cleucine label after incubation of myetomonocytic leukaemia cells with increasing adriamycin concentrations for 2 hours. The protein designated as no. 30 was selectively decreased in a dose-dependent fashion. This protein labelled well with 14C-leucine but, following 3~p-labelling data, is apparently not phosphorylated. In a comparative study no effects on NHP were noted following exposure of leukaemia cells to bleomycin (30 ~tg/ml) or actinomycin D (2.5 gg/ml).
Discussion
The extraction, separation and electrophoretic analysis of phenol-soluble acidic proteins associated with nuclear DNA has first been described by Teng et al. (1971). Most of these proteins have been shown to be phosphoproteins which incorporate 3Zp-orthophosphate in vivo leading to formation of phosphothreonine and phosphoserine residues. Teng et al. (1971) could demonstrate that a) the patterns of phosphorylation of individual proteins varied from tissue to tissue, b) binding of these proteins with DNA was species-specific, and c) some of these phosphoproteins stimulated transcription in a cell-free RNA-synthesizing system. Originally, this class of nuclear proteins was isolated from 0.32 M sucrose-3mM MgClz nuclei [36]. In our own studies on phenol-soluble proteins from various human leukaemia and lymphoma cells, a citric acid procedure was used since MacGillivray et al. (t972) had demonstrated that "sucrose nuclei" contained fewer high molecular weight species compared to "citric acid nuclei" in most tissues studied. Comparative analyses of phenol-soluble chromatin proteins in human leukocytes have not been reported in the literature. Very recently, two-dimensional gel electrophoresis studies of "Chromatin frac-
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tion II" proteins - a different protein fraction including more than 90% of the total chromatin-associated nonhistone proteins - were presented for normal lymphocytes, PHA-stimulated Iymphocytes and teukaemic lymphoblasts [44]. In these studies, some high molecular weight chromatin proteins were found increased in stimulated compared to normal lymphocytes. In addition, leukaemic lymphoblasts (ALL) were characterized by lower concentrations of individual fast moving proteins (A24, BA) and by enhanced protein concentrations in distinct spots of the high molecular weight region. However, a patient-to-patient variation was noted in these ALL patients which had been earlier observed in studies on acidic nuclear proteins from CML, CLL and AML cells (Weisenthal and Ruddon, 1972; 1973) probably related to protease activity. Nevertheless, these authors had demonstrated characteristic protein banding patterns in a comparison of different human leukaemic diseases [39]. It should be noted in this context that differences of chromatin proteins may be introduced by a different distribution of cells in the various stages of the cell cycle. Stein et al. (1975) have shown that HeLa S-phase chromatin contained a nonhistone protein which could render the histone genes available for transcription. This protein was not found in Gl-phase HeLa cells [33]. NHP changes as a function of various stages of the cell cycle have also been reported by other authors [20, 32]. Several conclusions may be drawn from the data presented in Figs. 1 to 6. It is apparent that the protein fraction analysed has some tissue specificity. The characteristic differences between normal lymphocytes and normal granulocytes with at least one specific protein (no. 25 in granulocytes, no. 26a in lymphocytes) have initiated further studies on the chemistry and function of these components. Protein no. 25 appears to be of special interest since it has been found in normal and leukaemic granulocytes but not in myelocytes (Fig. 5) or in lymphocytes. As both myelocytes and lymphocytes, under certain conditions, are able to divide, this fraction might represent some gene control protein (Fig. 5). The high number and concentration of high molecular weight proteins in the lymphosarcoma cells may reflect to some extent the high-grade neoplastic nature of these cells. In earlier comparisons of liver and hepatoma cells, a similar increase of high molecular nonhistone proteins has been described for highly malignant tumour cells [42, 43]. The labelling data from the experiment illustrated in Fig. 3 further suggest a relationship between the almost uniformly distributed phosphorylation of these phenol-soluble proteins and mitotic activities. The dose-dependent selective decrease of individual chromatin proteins after incubation with an inter-
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s. Seeber et at. : Nonhistone Chromatin Proteins in Normal and Leukaemic Human Leukocytes
c a l a t i n g a g e n t is a f i n d i n g o f special i n t e r e s t (Fig. 6). T h i s effect h a s also b e e n n o t i c e d in N o v i k o f f h e p a t o m a cells [30]. T h e d a t a i n d i c a t e t h a t a d r i a m y c i n a n d d a u n o m y c i n m a y exert a specific d a m a g e o n the n u c l e a r c h r o m a t i n o f v a r i o u s t u m o u r cells, d i f f e r e n t f r o m t h a t c a u s e d b y a c t i n o m y c i n D or b l e o m y c i n . T h e s t r u c t u r e o f the d a u n o m y c i n - D N A c o m p l e x h a s b e e n a n o b j e c t o f n u m e r o u s i n v e s t i g a t i o n s . O n the a s s u m p t i o n t h a t the m o d e l o f P i g r a m et at. (1972) is c o r r e c t - r e c e n t l y s u p p o r t e d b y D i M a r c o a n d A r c a m o n e (1975) - it c o u l d be c o n c l u d e d t h a t p r o t e i n no. 30, w h i c h was affected b y d a u n o m y c i n b o t h in the e x p e r i m e n t a l t u m o u r a n d the h u m a n l e u k a e m i a cells, s h o u l d be a m a r k e r p r o t e i n for t h e d a u n o m y c i n i n t e r c a l a t i o n site at the m a j o r g r o o v e of the D N A d o u b l e - h e l i x . Its d o s e - d e p e n d e n t decrease in p a t t e r n s o f tightly b o u n d , p h e n o l - s o l u b l e p r o t e i n s c o u l d be explained by a loosening of some DNA-protein bonds f o l l o w i n g d r u g i n t e r c a l a t i o n a n d local d i s t o r t i o n . T h u s , p r o t e i n no. 30 w o u l d be e l u t e d f r o m the c h r o m a t i n a l r e a d y at early steps o f the e x t r a c t i o n p r o c e dure. A n o t h e r e x p l a n a t i o n c o u l d be t h a t d u e to crosslinks o f d o u b l e - s t r a n d e d D N A a t the i n t e r c a l a t i o n site a n d d u e to a n a l t e r e d t e r t i a r y s t r u c t u r e o f the DNA, some proteins could become trapped. Both mechanisms would prevent individual proteins from e n t e r i n g t h e p h e n o l p h a s e f r o m w h i c h t h e final p r o t e i n f r a c t i o n was i s o l a t e d by dialysis.
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Received September 15, 1977 Accepted October 5, 1978 Priv.-Doz. Dr. S. Seeber Innere Universit~itsklinik und Poliklinik (Tumorforschung) Westdeutsches Tumorzentrum Hufelandstrage 55 D-4300 Essen Federal Republic of Germany
