In VitroCell. Dev. Biol. 26:857-864,September1990 9 1990Tissue Culture Association 0883-8364/90 $01.50+0.00

EXPRESSION

O F a- A N D /3-HUMAN C H O R I O N I C G O N A D O T R O P I N S U B U N I T S IN C U L T U R E D H U M A N C E L L S

SAMUEL GOLDSTEIN', RICHARD A. JONES, JAMES W. HARDIN, GLENN D. BRAUNSTEIN~, ANDROBERT J. SHMOOKLER REIS Departments of Medicine and Biochemistry & Molecular Biology. University of Arkansas/or Medical Sciences. and Geriatric Research, Education and Clinical Center, John L. McClellan Memorial Veterans Hospital. Little Rock. Arkansas 72205

(Received 17 January 1990; accepted 23 April 1990)

SUMMARY We surveyed several human cell lines for production of a- and /3-human chorionic gonadotropin (hCG~ under a variety of conditions known to induce gene expression, a- and/~-hCG subunits were monitored in culture media by specific radioimmunoassays and were shown to be quite sensitive to serum refeeding and growth state of all cell types studied. The permanent line JEG-3 secreted both a- and /~-subunits whereas HeLa cells secreted only the a-subunit. Production of both subunits was augmented in these permanent cell lines, for each growth state, by pretreating cells with 5-azacytidine; in contrast, spontaneous /3-hCG production by normal human fibroblasts (four of six strains) was only rarely increased after 5-azacytidine treatment, and more often was suppressed by 30 to 40%. Three of five strains from inherited chromosomal breakage syndromes produced immunoassayable 13-hCG spontaneously, two of which increased secretion upon treatment with either UV or mitomycin C. Surprisingly, one normal cell strain of fetal origin was induced to secrete a-hCG, but not/3-hCG, after UV irradiation. JEG-3 and HeLa cells produced detectable cognate mRNA for a- or/3-hCG subunits or both by Northern and S1 nuclease protection analyses, whereas such transcripts from untransformed human fibroblasts were consistently below detectable levels. Quantitation of /3-hCG mRNA by R N A : R N A annealing kinetics indicates that even the fibroblast strain producing the highest secreted /~-hCG levels contained cognate mRNAs at only ~0.1 copy per cell. We conclude that hCG expression in human fibroblasts is strongly repressed at the transcriptional level, although a variety of conditions igrowth state, serum refeeding, cell senescence, or DNA damage} can affect the level of "leaky" expression, at least in some responding fraction of cells. K e y words: chorionic gonadotropin; ectopic gene expression; genetic repression; cell culture.

INTRODUCTION

Fishel et al., 1984). The a-subunit of this gene, common to a number of peptide hormones, is frequently activated in a variety of malignant tissues producing "ectopic" hormones {Vaitukaitis, 1979; see Braunstein, 1983), and seems to be expressed at low levels in several normal adult tissues (Braunstein, 1982). Although the a-subunit locus is present in a single copy within the human genome (Fiddes and Goodman, 1981), the fi-hCG subunit is present in six gene copies, of which only two may be actively expressed (Policastro et al., 1986). Surveys of human fibroblasts by sensitive radioimmunoassay have indicated that low-level fl-hCG expression occurred in a total of 11 of 40 fibroblast strains, whereas only 1 of 40 produced detectable a-hCG subunit (Rosen et al., 1980; Milsted et al., 1982). Cultured human fibroblasts, therefore, seem to be variably "leaky" at the fi-hCG locus and essentially repressed at the a locus, despite the loss of methylations in this latter gene during serial passage (Shmookler Reis and Goldstein, 1982a,b; Goldstein and Shmookler Reis, 1985; Shmookler Reis et

Regulation of gene expression in eukaryotic cells may occur at several levels in a multistep sequence. In the case of highlyspecialized genes, whose specific mRNAs may account for a large proportion of the total, it seems that control is exerted primarily at the level of transcription {Nevins, 1983}. In contrast, the great majority of genes in a given cell type are essentially completely repressed (Davidson, 1976), while those genes that are expressed are substantially attenuated at the transcriptional level and further modulated posttranscriptionally ( Kozak, 1988 ). Human chorionic gonadotropin (hCG) is abundantly expressed in first trimester placenta as well as in a variety of other tissues early in life IMcGregor et al., 1983; ' To whom correspondence should be addressed at John L. McClellan Memorial Veterans Hospital, 151 Research, Little Rock, AR 72205. Present address: Department of Medicine, Cedars-Sinai Medical Center and UCLA School of Medicine, Los Angeles, California 90048. 857

858

GOLDSTEIN ET AL.

al., 1989). Factors that might distinguish leaky from nonleaky cell strains remain to be determined. We here report the results of a broad comparative survey, intended to examine the effect on hCG subunit expression tif any) of cellular senescence, growth state, genetic instability, and specific treatments known to induce global hyp0methylation or D N A damage.

MATERIALS AND METHODS

Materials. Eagle's minimum essential medium was purchased as the dry powder (GIBCO, Grand Island, NY) and reconstituted in our laboratory. Sera were purchased from Hyclone (Logan, U T ) or Whittaker (Walkersville, MD), after pretesting for maximum growth potential. Restriction endonucleases and other enzymes were purchased from Bethesda Research Laboratories (Rockville, MD), New England Biolabs (Beverly, MA), and Pharmacia (Piscataway, N J). Standards and antisera for radioimmunoassay of a- and fl-hCG were provided by the Center for Population Research, National Institute of Child Health and Human Development, Bethesda, MD. Radiolabeled materials were purchased from Amersham Corp. (Arlington Heights, IL) or New England Nuclear (Boston, MA). Nitrocellulose filters (BA85) were from Schleicher and Schuell (Keene, NH ). All other reagents and chemicals were of the highest purity available and were obtained from Sigma (St. Louis, MO), Calbiochem (San Diego, CA), or Pharmacia. Sodium butyrate was prepared as a 0.5 M stock by titrating n-butyric acid with NaOH to a pH of 7.2. Cell culture. The sources and replicative life spans of human fibroblast strains in our survey are given in Table 1. Three of these strains (PKR, 376T, and T82) had previously been shown to secrete ~-hCG-like material (refs. in Table 1). Cells were grown in 100-mm petri dishes or 850-cm 2 roller bottles (Corning, Corning, NY), depending

on the bulk needed for R N A preparations. The HeLa $3 subline was obtained from Dr. S. Kit ~Baylor College of Medicine, Houston, TX) and JEG-3 cells tKohler and Bridson, 1976; Burnside et al., 1985) were obtained from Dr. P. Kohler tUniversity of Arkansas for Medical Sciences, Little Rock, ARh fibroblast strains from the chromosomal breakage syndromes were provided by the Genetic Mutant Repository, Camden, NJ. Fibroblasts were propagated routinely in Eagle's minimum essential medium supplemented with 15% calf serum; serum was reduced to 10% for HeLa and JEG-3 cells. Cells were incubated in a 95% air:5% CO2 humidified atmosphere at 37 ~ C. Cells were treated with various chemical agents and with UV light in specific protocols described in legends. In treatments with 5-azacytidine (4 ~M), cells were exposed to this agent for 48 h during logarithmic ~logl growth. After rinsing with phosphate buffered saline (PBS} and refeeding with fresh growth medium, cells were allowed to grow to confluence when they were harvested for R N A preparation (see below). Details of other chemical and UV treatments are given in the text or legends. Radioimmunoassay [or a- and fl-hCG. Inasmuch as it has been demonstrated that/>90% of the a- and fl-hCG produced by cultured cells is secreted into the medium tRosen et al., 1980; Peters et al., 1984), we conducted all assays on culture medium that had been exposed to cells. Background samples consisted of naive growth medium. The a- and fl-subunits of hCG were measured with previously described radioimmunoassays (Braunstein et al., 1978; Braunstein et al., 1979) with slight modifications that include reduction of the assay volume to 0.5 ml and dilution of the standards in fresh growth medium. The sensitivity of the a-subunit assay was 0.5 ng/ml of medium, and the intraassay coefficient of variation and interassay coefficient of variation were 12.3 and 13.6%, respectively. Assay for the p-subunit had a sensitivity of 0.4 ng/ml, intraassay coefficient of variation of 8.7 to 11.4% and interassay coefficient of variation of 8.8 to 15.7%. DNA probe. The a-hCG probe was the 440 base pair (bp) {PstI fragment of the cDNA provided by Dr. I. Boime

TABLE 1 CULTURED HUMAN FIBROBLASTS SURVEYED FOR hCG PRODUCTION Replicative Life Span, MPI)"

Strain

Age ~YearsL Sex of Donor

Tissue Source

Normal HSC172 PKR 376T A2 T82 J088

fetus, F newborn, M 7, M 11, M 58, M 76, F

lung foreskin skin skin skin skin

70 55 ND 60 ND 50

Goldstein and Shmookler Reis, 1985 Milsted et al., 1982 Rosen et al., 1980 Harley and Goldstein, 1978 Milsted et al., 1982 Goldstein et al., 1979

Chromosomal breakage syndromes" BS GM2548 FA GM0369 FA GM2053 XPA GM0554 XPA GM0882

6, M 6, M 12, F 10, M 12, F

skin skin skin skin skin

ND ND ND ND ND

NIGM HGMCR' NIGM HGMCR NIGM HGMCR NIGM HGMCR NIGM HGMCR

|~.eferenee

"Cumulative number of mean population doublings at replicative senescence. bBS: Bloom syndrome; FA: Fanconi anemia; XPA: xeroderma pigmentosum complementation group A. ~National Institute of General Medical Sciences, Human Genetic Mutant Cell Repository, Camden, NJ. determined; MPD = mean population doublings.

ND = not

hCG EXPRESSION IN CULTURED CELLS

(Washington University School of Medicine, St.Louis, MO) (Boothby et al., 1981). The/3-hCG probe, from a plasmid supplied by Dr. J. Fiddes (California Biotechnology, Inc., Mountain View, CA), was the 579 bp c D N A tFiddes and Goodman, 1980). Preparation of RNA. Cultured ceils were rinsed with PBS and harvested by adding guanidine thiocyanate to roller bottles and scraping with a rubber policeman, as described by Chirgwin et al. (1979L followed by CsCI centrifugation. PolyA § R N A was prepared from total cellular R N A by two cycles of oligo dT chromatography (Maniatis et al., 1982).

S1 nuclease protection

assay for RNA

transcripts.

Analysis of a-hCG transcripts was carried out as described by Favoloro et al. (1980). For the a-hCG probe in pBR322, the plasmid was linearized with X b a I at a single site in the a-hCG cDNA. After kinase labeling with r-32p-ATP, and cutting at the H i n d I I I site in pBR322, fragments were gel purified. The fragment containing the anti-sense strand, that produced an S1 nuclease-protected R N A band of 274 nt after annealing with placental polyA§ RNA, was used in subsequent assays. The/3-hCG cDNA, which was inserted in the H i n d I I I site of pBR322, was cut with PvuII, kinase labeled with y-32P-ATP, followed by H i n d I I I digestion. The S1 nuclease-protected fragment obtained with placental R N A was 345 nt.

859

determined on replicate dishes. Figure 1 A shows that the peak rate of specific /3-hCG production (as a fraction of total cell protein) occurred between 4 and 7 d, as cells became densely confluent. Treatment of cells with 5-azacytidine did not significantly alter /3-hCG production during this growth period. In the second protocol, where cells were grown without refeeding until Day 7 (Fig. 1 B), peak production of /3-hCG occurred around Day 4, at which time cells were approximately half confluent. Medium was replenished after 1 wk, as cultures became confluent, and a second peak of fl-hCG output was seen 2 d later (Day 9). It should be noted that specific fl-hCG production per day is markedly reduced in the absence of frequent refeeding (Fig. 1 B), relative to cells receiving daily mitogenic stimulation with serum (Fig. 1 A). Treatment with 5-azaC was somewhat inhibitory to fl-hCG production (possibly due to toxic or growth-inhibitory effects), although it seemed to elicit a more rapid rise in fl-hCG production by early-log cells (Day 2).

A

]

.........

* - - CO~.,UEI~'CE - - - *

[~---I,OG OROWTH ----~

,o

o ~

6~

o

Analysis of low level transcripts by reassociation kinetics. R N A probe labeled either with 35S-UTP or ~2P-UTP was prepared in the Gemini system (Melton et al., 1984) using SP6 R N A polymerase. Labeled RNA, either a-hCG or fl-hCG antisense strand, was incubated as described by Mathews et al. (1986) in a 40-/al vol containing 40 m M piperazine-N,N'bis[2-ethane sulfonic acid] buffer, pH 6.7, 0.5 M NaCI, 1 m M E D T A , 25~ formamide, and variable amounts of polyA§ R N A from the anti-sense standard, the placental R N A standard, or from cultured cells plus yeast t R N A to achieve a constant 40 #g of total R N A in 32/al. The reaction mix was covered with paraffin oil to prevent evaporation, denatured at 85 ~ C for 5 min, and hybridized for 18 h at 70 ~ C. This temperature was determined to give maximal annealing over the range of 50 to 80 ~ C. After the addition of 100 /ag denatured herring sperm DNA, samples were digested with RNase A and RNase T1 at 37 ~ C for 1 h. Preliminary studies determined the RNase concentrations and times of digestion to optimize the signal-to-noise ratio. RNase-treated samples and undigested controls were then precipitated with 10% cold trichloracetic acid, collected on G F C filters, rinsed, and dried, followed by determination of acid-insoluble counts in a liquid scintillation counter. RESULTS

Dependence of fl-hCG secretion on growth conditions for T82 fibroblasts. Studies were first carried out on T82 fibroblasts, known to produce /~-hCG ectopically (Milsted et al., 1982), to determine the influence of culture conditions and growth state on fl-hCG production. Cells were split at a 1:2 ratio into 100-mm petri dishes and examined in two protocols. In the first, the medium was completely replaced on a frequent schedule. Radioimmunoassays were done on each sample of the medium used for cell culture, whereas protein content was

0

1

2

4

3

5

6

7

DAYS AFTER PLATING

B LOG GROWTH

q

CONFLUENCE

o

/\ o

r~

~

1-

.

,.~ I Q~"

A

.

x x

O[ 0

2

i

; DAYS

;

,'o

t~

AFTER PLATING

FIG. 1. Production of fl-hCG in T82 human fibroblasts during different refeeding schedules. A, T82 fibroblasts were subcultured at a 1:2 split ratio into 100-mm petri dishes at time 0. Medium H0 ml/dish) was replaced each day for the first 4 d and on Day 7. Radioimmunoassays were carried out on each sample of spent medium, with protein determinations carried out on replicate dishes. Results are expressed as the increment in fl-hCG production'24 h-"total mg-~ of protein in each dish on each day of measurement. Open circles = minus 5-azaC; open triangles = plus 5-azaC. B, T82 ceils were split at a 1:2 ratio and grown without replenishment of medium until Day 7, when the medium was changed. Each dish initially contained 15 ml of growth medium with sampling of 1 ml each day for radioimmunoassay. Protein content was determined on replicate dishes and results expressed as the increment in fl-hCG production" d-' "total mg-' protein. Symbols as in A.

860

GOLDSTEIN ET AL.

Effects of growth state, replicative senescence, and 5-azaC for various cell lines and strains. A v a r i e t y of

s u r v e y e d for r a d i o i m m u n o a s s a y a b l e a- a n d / ~ - h C G secret i o n as a f u n c t i o n of g r o w t h s t a t e a n d 5 - a z a C t r e a t m e n t

cultured

{ T a b l e 2). S a m p l e s w e r e o b t a i n e d e i t h e r a t D a y s 3 to 4 [log {L)

human

cells,

described

in

Table

1,

were

TABLE 2 P R O D U C T I O N OF h C G S U B U N I T S {ng" mg-' P R O T E I N " d-'} Alter 5-Azacytidine Growth State"

a

fl

a

JEG-3

L C 24 ~ pc

135.7 142.9 43.9

73.1 44.9 0.7

576.8 {X4.21 403.9 ~X2.8~ 633.2 {X14.4J

163.5 {X2.2J 293.3 {X6.5} 799.1 {X1140J

HeLa

L C 24 ~ pc

10.1 30.1 21.0

< <