Drug modulation of chromosomal protein subtypes during specific phases of the submaxillary cell cycle

Cell Biology International Reports, Vol. 8, No. 4, April 1984 279

DRUG MODULATION OF CHRCNOSOMAL PROTEIN SUBTYPES WRING SPECIFIC PHASES OF THE SUBEIAXILLARY CELL CYCLE

*Pipkin, J.L., Anson, J.F., Hinson, W.G., and **Burns, E.R., NCTR, HFT-164, Jefferson, Arkansas 72079; **UAMS, Department of

Anatomy, Little Rock, Arkansas 72205 (*Reprint requests)

ABSTRACT The Hl subtype proteins a, b, c, d, e, and lo and the high

mobility group (HMG) proteins 14 and 17 were extracted from submaxillary gland (SK) nuclei treated in vivo and sorted from -- specific phases of the cell cycle, and analyzed by gel electrophoresis. Significant differences in these proteins were noted between quiescent (G ) phase nuclei and proliferating nuclei in both stained and aut radiographic gels. 8 . Stimulation of the SK into cell division with DL-isoproterenol-HCl alone or in conjunc- tion with sodium phenobarbital (PB) pfrovided a analysis of drug effects in stained,

syst% for the H lysine, and P pulse-

labeled nuclear proteins obtained from different phases of the cell cycle. INTF0DUCTION

The use of isoproterenol (IPR) for initiating ordinarily quiescent epithelial tissues into proliferation provides an excellent method for examining drug effects upon nuclear protein synthesis and LMA replication (Pipkin et al., 1981, Pipkin et --

., 198213). -

&., 19822, Pipkin et al Several subtypes of nuclear proteins (Bl histones and high

mobility group proteins-B.%) are possible biological indicators of toxic stress since alteration in their synthesis and phosphorylation patterns are extremely sensitive to changing conditions (Lennox et al., 1982; Bhorjee, 1981).

The Hl hi%o%s are ccmposed of multiple subtypes, usually three to six forms, varying in nurrber of subtms which correlate with tissue types and states (Lennox et &., 1982). In our attempt to identify tiological indicator proteins of chemical stress, we have discovered that drug administration alters the Ii proteins. In this work, the H1 proteins reflect a quiesccn t state in their subtype forms in the mature SKG; however, after administration of IPR and PB the Bl subtype forms are characteristic of embryonic or juvenile tissues.

The M!G proteins (14 and 17) are associated with regions in the chrcmatin that contain active or potentially active gene sequences (\:eisbrod et g., 1380, Sandee et al., 1980). lie observe that BM3 17 and especially 14 reveals increased phor- phorylation at specific tires in the cell cycle after drug administration.

The evidence suggests that synthesis and phosphorylation of the I:, subtypes (Lennox et al., 1982) and the HM3s (Seyedin et g. I t979) are either temporarily or directly correlated wi?% structural and/or regulator functions of the cell. These facts have prompted us to examine the effect of human therapeutic drugs on the n:etat;olism and synthesis of tissue during the in vivo cell -- cycle.

0309-1651/84/040279-09/$03.00/O @ 1984 Academic Press Inc. (London) Ltd.

280 Cell Biology International Reports, Vol. 8, No. 4, April 1984

MATERIALS ANDMHTHODS Rats of the CD strain, 6-8 weeks of age, were injected with

a single dose of 25 mg/lOO g body weight DL-isoproterenol-HCl (Sigma Chemical Co. St. Louis, Mo) (Pipkin et &., 1980), a single dose of 5.0 q/100 g bocQ weight of PH (Mallinckrodt, Inc. Paris, KY) (Watson et al., 1975) or a combination of the two. At -7. 28 hr after drug administration, when the sutznaxillary gland yndergces the highest rate of DNA synthesis (Burns, 1978), either 39-lysine at 0.25 r&i/l50 g body weight (NEN, Boston, MA) or

P-orthophosphoric acid at 2 mCi/lSO g body weight (NHN, Boston, MA) in 0.5 ml normal saline was injected IP follmed 2 hr later by a "chase" of ncn-radioactive lysine or phosphate in saline. Animals were sacrificed 30 min after "chase" and the submaxillary gland removed and frozen on dry ice.

Submaxillary gland nuclei were purified (Urban et al., 1979) stained with propidiun iodide (Sigma Chemical, St. Lziz MI)) and sorted on the basis of DNA fluorescence using a fluorescence activated cell sorter (FACS II Pecton-Dickinson) (Herzenberg et al., 1976) directly into specially designed collection chambe= (Hinson et al., 1981). -- The nuclear populations sorted for study include: the control (non-stimulated) G (2C) peak: IPR stimlated mid Gl to mid S [Gl(2C)+S] peak, a& mid S to late G /M [S + G (4C)l peak, PB treated (non-stimlated) G (PB) pea , and is the2combination IPR + PB stimlated Gl + S (IPR 0 PB) peak, and S + G2/M (IPR + PB) peak.

The sorted nuclear sqles of 5 to 10 million each were cen- trifuged and the air-fuge tubes containing the resulting nuclear pellet were removed fram the McLecd ch&rs (Hinson, et al., 1981). -7 Ihe nuclek were suspended in the appropriate extracting solution (10 pl/lO nuclei) (Pipkin et al., 1982b) and disrupted by sonication (Heat Systems Ultra&i= Model W200R with 420 microtip, Plainview NY). The H histones ware extracted directly from the disrupted nuclei wi h *Ii 5% (w/v) perchloric acid and precipitated with trichloroacetic acid 20% (w/v) after the procedure of Johns and Butler (1962) as modified by Lennox and Cohen (1983). The HMGs were extracted from sorted nuclei by the procedure of Goodwin et al., (1973) omitting the filtration process. The san@esweE ultracentrifuged in an Air Fuge (Beckman Instruments, Palo Alto, CA), and the purified protein obtained was dialyzed against electrophoretic electrolyte (Laemnli, 1970) in an ultra-microdialysis apparatus (Pipkin et - &., 1982b).

Electrophoresis of tha H histones in polyacrylamide gel was mcdeled after the procedure o 4 Lennox et al. (1982), using acid- -- urea in the first-dimension and sodiun dcdecyl sulfate (SDS) in the second-dimension. The HMG proteins were examined electro- phoretically by single-dinension SDS slab gels (Laemmli, 1970). Staining was with Coomassie brilliant blue R-250 (Fairbanks et - al., 1971). -

Gel. autoradiography was done according to ths procedure of Bonner and Laskey (1974). Detection, excision, and elution of the proteins from the gels was done according to the procedure of


Pipkin et al. (1980). -- Quantitative ccsrparisons of proteins were done by area

measurements of gel spots using a Videoplan Version 4 (Carl Zeiss, Inc.). RESULTS

Staininq of the Hl Subtypes During the Cell Cycle The Hl proteins of the quiescent SMG (G phase) demonstrated

limited staining of the subtypes 'a' and '8, after examination & two-dimensional gel chromatography (Fig. l,A).

b d b

a a C

f

A D lo

P

Figure 1. Chromatographic and Autoradiographic (3H-lysine) Patterns of Hl Subtype Proteins Various Phases of the Cell Cycle Run in Two-Dimensional Slab Gels.

A. Go (control) stain; B. G + S (IPR) stain; C. G ?I-lysine;

+S (IPR+ PB) stain: D. G (confrol) F. Gl + S (IPR 9 PB) H-lys?ne

E. Gl + S (IPR13H-lysine);

120 vg of protein was used in the stained chrcmatograms, and 200 pg of protein was used in the autoradiograms.

282 Ceil Biology International Reports, Vol. 8, No. 4, April 7984

Subtypes 'C', 'd' and 'e' stained prominently with 'e‘ being the major contributor, asoshm by area spot measurements (Fig. 1,A; Table 1). Subtype '1 ' was possibly conposed of two ccsponents.

Ihe administration of IPR initiated proliferation of the otherwise quiescent SMG during G increase in subtypes 'a', 'b', '&I

+ S phases with a ccncomitant and 'd' (Fig. 1,B; Table 1).

TABLE1

Hl Subtypes Area Measurements of Stained Hl Subtype Proteins

Mean + Standard Deviation

Hl Subtypes Stain (Comnassie Brilliant Blue)

GO Control Gl-S IPR Gl-S IPR + PB

a

b C

d

e

lo

Complex

15+ 6 - 23 + 13 71 f. 17

71+ 23 - 186 213

105 14 +

269 216

(d + e)

66+ 7

128 + 13 - 89+ 4 - 96 + 13 *

362 4

44 + 10

270 + 14 - (b + d + e)

76+ 5 - 133 + 21 - 109 + 5 - 1062 5

232 2

15+ 5 - 271 + 19 -

(b + d + e)

Area measurements of the H subtypes were made with a Videoplan (Carl Ziess, Inc.). Measu emants were taken from three separate h chromatqraphs and standard deviations were calculated.

Increased staining in subtypes 'a' and 'b' was especially dramatic. Subtypes 'e' and '1 ' were significantly reduced after IPR administration.

After dosing with IPR and PB, an increase in the staining areas of 'a' and 'c' occurred with slight reduction in 'e' and a significant reduction in 'lo' (Fig. 1,C; Table 1).

3H-Lysine Incorporation of Hl Subtype During the Cell Cycle The six Hl protein subtypes 05 the quiescent Sffi (G

revealed limited incorporation of H-lysine after pulse P phase)

abaling by two-dimensional autoradiography (Fig. 1,D). greatest in subtype 'e' and faintly visible in 'lo'.

Labeling was

After the administration of IPR, there was a substantial increase in labeling of most subtypes, especially of 'a','b','c', and 'd' (Fig. 1, E). Subtype 'e' increased tilt was still weakly labeled. It was difficult to assess the labeling of '1".

Cell Biology international Reports, Vol. 8, No. 4, April 1984 283

Dosing with IPR and PB of the Gl + S phase produced radioactive patterns of Hl subtypes very similar to stained patterns treated under the same conditions (Fig. l,F). For example , subtype 'a' and 'c' showed increased amounts of incorporation when compared with Sffi dosed solely with IPR. The inclusion of W with IPR resulted in the highest incorporation of 'c'.

Phosphorylation of the Hl Subtype Proteins The H

a chromosomal pro3 ins

% from IPR-stimulated SMG

demonstrate phosphorylation ( ) of ten distinct subtypes & two-dimensional gel autoradicgraphy (Fig. 2,A).

Figure 2. Phosphorylation (32P) Patterns of H1 Subtypes Protein from G2 Phase of the Cell Cycle Run in Two-Dimensional Slab Gels.

A. G2 (IPR) B. G2 (IPR + PB)

120 Lg of protein was used in each autoradiqram.

After the inclusion of PB in the dosing regime, additional phosphorylation modification occurred in spot 2 and in the region of subtypes 9 and 11 (Fig. 2,B). EEe have attempted to identify the phosphorylated H subtypes according to the nomenclature of Lennox et al., 1982, &ut -- differences may exist. The control (Go) and PB-treated sartples revealed faint phosphorylation patterns and were ooorlv resolved (not shcwn).

Phosphoryiation of the Hl and HMG,$roteins Relatively lm phosohorvlation ('-P) of the H, and HMG 14

and 17 chrc~~&ca~l proteins-of the quiescent SMG 'was shm by single dimensional gel autoradiography (Fig.3, lane 1). After administration of IPR, a slight increase in phosphorylation was observed in H

4

and HMG 17 during G phase of the cell cycle (lane 2). During phase, IPR-stimula ion produced additional phos- k phorylation o the HMGs with 14 showing the greatest increase (lane 3). The administration of PB produced phosphorylation patterns similar to the control (lane 4). The combined dosing of IPR and PB enhanced phosphorylation of H1 in Gl (lane 5) with

284 Cell Biology international Reports, Vol. 8, No. 4, April 7984

increased phosphorylation of H in G (lane 6). The most prti- nent effect of the combined ladmin?stration of IPR and PB on phosphorylation of the HMGs was the intense labeling of 14.

Figure 3. Phosphorylation (32P) Patterns of H Histone and HMG (14 and 17) Proteins fran Various Phases of the'Cel1 Cycle Run in Single-Dimensional Slab Gel.

Lane: 1. GO (control) 2. G1 + S (IPR) 3. G2 (IPR) 4.Go (PB) 5. G + S (IPR + PB) 6. G1 (IPR + PB) 7. Sgained profile of carbonic anhydrase (30KDa) and

lysozyme (14KDa)

30 llg protein was introduced in each lane.

DISCUSSION The phosphorylation states of HMG 14 and 17 can be corre-

lated with stage-specific changes in gene expression. It is possible that increased phosphorylation of Hffi 14 is a pre- requisite for the process of chromatin condensation and cyto- kinesis occurring after the G2 phase of the cell cycle (Bhorjee, 1981). There is as much circumstantial evidence for the functional role of H1 in gene expression as for the HMGs. The H possibly functions in regulation & a mechanism of conformationa i alterations of chrcaretin (Ajiro. et al., 1981a; Ajiro.s &., -- 1981b). Additicnal evidence for the H1 regulatory capacity stems from their tissue specificity (Panyim et al., 1981; Lennox et g. I 1983), -? alterations associated with tissue differentiation (Stellwagen and Cole, 1969; Lennox et al., 1983) and changes -- during anbrycnic development (Ruderman and Gross, 1974).

The numerous modifications of the HMGs and Hl subtypes associated with various tissue states are extremely intriguing


and bring strong evidence to bear for thair functional role in gene expression; hcwever, 1x3 information exists linking alteration of these subtypes with enviromntal shifts evoked by drug or toxic chemical exposure. & have shown in this work that alteration in synthesis and phosphorylation of these subtypes can be accomplished & the administration of drugs.

In catparing our data with results of Lennox and Cohen (1983), we find that the Hl subtypes of SMG from 8 week old rats possess staining and autoradiographic patterns reminiscent of quiescent or aged tissue. The non-proliferating state of the SK is characterized by extremely lcw amounts of 'c' and 'd' and an abundance of 'e'. Administration of a single dose of IPR stimu- lates synthesis and isotope it-corporation of certain Hl subtypes and the reduction of these events in others. The increase in subtypes 'a' an$,'b' during Gl + S is dramatic and the reduction in 'e' and '1 is equally as spectacular. Ths reciprocal relationship of subtypes 'a','b' and 'c' with '1 ' in their staining and labeling during the cell cycle is noteworthy. Subtypes 'a', 'b', and 'c' incregse and subtype 'lo' decreases. The increase in staining of '1 ' has been correlated with a decrease in tissue netabolisn caused & artificially inducing quiesence (Seyedin et al., 1981) or @ natural aging (Lennox and Cohen, 1983). In this present study, the administration of IPR is accompanied by an increase of DNA synthesis in the SMG as shown in other studies, (Pipkin et &., 1980; Pipkin et al., 1982b) followed t.&~ a several fold reduction in 'e' and 'l-.-In our past mrk, stirrollation of DNA replication by IPR (Pipkin et al., 1981; Pipkin et al., 1982a, Pipkin et al., 1984) was never -- - -. associated with a reduction in protein synthesis. of 'e' and 'lo'

The reduction is the first observation by us of the repression

of synthesis and labeling of major protein complex(s) associated with the administration of this drug.

The additive effect of PB in enhancing 3 H-lysine incorporation of spot 'c' and phosphorylation of spots 2 and 9 in IPR- treated animals, reflects the sensitivity of these subtypes to drug administration. It is interesting that PB had no effect upon synthesis or isotope incorporation of the Hl subtypes unless given in ccmbination with IPR. This synergistic effect of PB in IPR-stirmrlated tissue is also observed in the HMG subtypes, where there is increased phosphorylation of Hm; 14 and 17 during the G phase. It is unclear at this tima what proportion of isot& incorporation reflects protein synthesis and what proportion represents protein modification.

The practical use of the H metabolic shifts frcm chemical e ho

subtypes as indicators of sure,

possibilities, may possess exciting

and awaits further indepth study with other organs and conponds. The stimulating effect of IPR on epithelial organs, the sensitivity of these subtypes to change, and ti-ra resolution achieved with gel chromatography and autoradiography demonstrates the potentiality of these techniques for the investigation of toxic stress in the in vivo system. --

286 Cell Biology International Reports, Vol. 8, No. 4, April 1984

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-'

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Revised version accepted: Received: 30th September 1983. 9th March 1984

Documents

Drug modulation of chromosomal protein subtypes during specific phases of the submaxillary cell cycle