IN VITRO Volume 14, No. 9, 1978 All rights reserved 9
R E G U L A T I O N OF T H E S Y N T H E S I S OF H U M A N C H O R I O N I C G O N A D O T R O P I N BY S T R A I N S O F H E L A C E L L S IN C U L T U R E JANICE YANGCHOU Section on DevelopmentalEnzymology, Laboratoryof BiomedicalSciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 SUMMARY
Thirty-seven strains of HeLa cells were examined for their ability to synthesize human chorionic gonadotropin (hCG) and its alpha subunit (hCG-a) in culture. Synthesis of hCGa and hCG also was investigated in the presence of sodium butyrate and 5-bromo-2'-deoxyuridine (BrdUrd). All HeLa strains synthesized hCG-a in culture. Sodium butyrate increased the synthesis of hCG-a in all HeLa cells; BrdUrd increased synthesis in 32 of the 37 strains examined. Although few HeLa strains synthesized hCG in the absence of inducers, hCG was detected in most strains in the presence of sodium butyrate. The synthesis of hCG and its alpha subunit is, therefore, a stable genetic characteristic of HeLa cells. Key words: hCG; HeLa cells; sodium butyrate; 5-bromo-2'-dcoxyuridine. INTRODUCTION Many human cell lines in culture have been shown to be of HeLa origin by karyotypes and by genetic and biochemical evidence (1-4L HeLa, the first established human cell line, was derived from a cancer of the cervix (5). HeLa cells in culture are characterized by their peculiar chromosomal markers (2, 3, 6-9), their lack of the Y chromosome (10), and their synthesis of A-type glucose-6-phosphate dehydrogenase (1). It has been demonstrated recently that certain HeLa strains secrete human chorionic gonadotropin (hCG) Ill), its alpha subunit (hCG-a) (12}, or both (13-15). HCG and several other human glycoprotein trophic hormones, including thyroidstimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH), are composed of two nonidentical subunits, alpha and beta {16-19). These four hormones have virtually identical a-subunits; in contrast, each has a unique/3-subunit which is responsible for biological specificity (18). Various HeLa strains have appeared during inadvertent isolation and have been cultured under variable laboratory conditions; they have been identified as HeLa by their chromosomal structures and their specific isoenzyme patterns. Since the secretion of hCG-a and hCG are characteristic of certain known HeLa strains, other strains thought to be HeLa also should secrete these proteins. Data given here demonstrate that each of 37
HeLa strains (4, 20) synthesized hCG-a and, in many cases, hCG in culture. MATERIALSANDMETHODS Cells and method of culture. All cells were grown in a-modified minimal essential medium. Monolayer cultures in 25-cm2 flasks were incubated at 37~ C in 5 ml of medium supplemented with 10% fetal bovine serum, streptomycin (100 pg per ml), and penicillin (100 U per mlk Medium and serum were obtained from Flow Laboratories, Rockville, Maryland. The medium was replaced every 2 days. Approximately l0 s cells were seeded in each of the 37 cultures. Cultures in the midexponential growth phase were used in this study because the amount of hormone produced per cell is relatively constant during this period. Cell lines used were obtained either from Dr. W. Nelson-Rees, Dr. G. Melnykovych, or from the American Type Culture Collection (ATCC). Radioimmunoassays and radioreceptor assay. A specific double-antibody radioimmunoassay using hCG and hCG-a as reference standards was used to measure hCG and hCG-a in the culture medium (21). Antisera used were: anti-hCG-f~ (Sb6) for hCG (22), and anti-hCG-a (CA3) for hCG-a {14). Purified preparations of hCG (CRl19; 11,600 IU per rag; ventral prostate weight assay; Second International Standards hCG) and of hCG-a (CR117) were radioiodinated and used as standards and tracers. HCG (CRl19) had 1% to 3% cross-reactivity in the hCG-a as-
775
CHOU
776
TABLE 1 EFFECT OF SODIUMBUTYRATEANDBRDURD ON HCG-t~ ANDHCG SECRETIONa
Strain
Control
hCG-a
hCG
Sodium butyrate
Sodium butyrate
ng / mg protein 82 1 49 124 207 448 917 2,940 2,778 2,450 93 253 560 437 837 550 2,270 926 1,648 13,830
BrdUrd
Control
BrdUrd
ng / mg protein 6.9 114.8 5.9 1,556.0 52.3 69.0 136.7 207.1 303.0 33.3
R E G U L A T I O N OF T H E S Y N T H E S I S OF H U M A N C H O R I O N I C G O N A D O T R O P I N BY S T R A I N S O F H E L A C E L L S IN C U L T U R E JANICE YANGCHOU Section on DevelopmentalEnzymology, Laboratoryof BiomedicalSciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 SUMMARY
Thirty-seven strains of HeLa cells were examined for their ability to synthesize human chorionic gonadotropin (hCG) and its alpha subunit (hCG-a) in culture. Synthesis of hCGa and hCG also was investigated in the presence of sodium butyrate and 5-bromo-2'-deoxyuridine (BrdUrd). All HeLa strains synthesized hCG-a in culture. Sodium butyrate increased the synthesis of hCG-a in all HeLa cells; BrdUrd increased synthesis in 32 of the 37 strains examined. Although few HeLa strains synthesized hCG in the absence of inducers, hCG was detected in most strains in the presence of sodium butyrate. The synthesis of hCG and its alpha subunit is, therefore, a stable genetic characteristic of HeLa cells. Key words: hCG; HeLa cells; sodium butyrate; 5-bromo-2'-dcoxyuridine. INTRODUCTION Many human cell lines in culture have been shown to be of HeLa origin by karyotypes and by genetic and biochemical evidence (1-4L HeLa, the first established human cell line, was derived from a cancer of the cervix (5). HeLa cells in culture are characterized by their peculiar chromosomal markers (2, 3, 6-9), their lack of the Y chromosome (10), and their synthesis of A-type glucose-6-phosphate dehydrogenase (1). It has been demonstrated recently that certain HeLa strains secrete human chorionic gonadotropin (hCG) Ill), its alpha subunit (hCG-a) (12}, or both (13-15). HCG and several other human glycoprotein trophic hormones, including thyroidstimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH), are composed of two nonidentical subunits, alpha and beta {16-19). These four hormones have virtually identical a-subunits; in contrast, each has a unique/3-subunit which is responsible for biological specificity (18). Various HeLa strains have appeared during inadvertent isolation and have been cultured under variable laboratory conditions; they have been identified as HeLa by their chromosomal structures and their specific isoenzyme patterns. Since the secretion of hCG-a and hCG are characteristic of certain known HeLa strains, other strains thought to be HeLa also should secrete these proteins. Data given here demonstrate that each of 37
HeLa strains (4, 20) synthesized hCG-a and, in many cases, hCG in culture. MATERIALSANDMETHODS Cells and method of culture. All cells were grown in a-modified minimal essential medium. Monolayer cultures in 25-cm2 flasks were incubated at 37~ C in 5 ml of medium supplemented with 10% fetal bovine serum, streptomycin (100 pg per ml), and penicillin (100 U per mlk Medium and serum were obtained from Flow Laboratories, Rockville, Maryland. The medium was replaced every 2 days. Approximately l0 s cells were seeded in each of the 37 cultures. Cultures in the midexponential growth phase were used in this study because the amount of hormone produced per cell is relatively constant during this period. Cell lines used were obtained either from Dr. W. Nelson-Rees, Dr. G. Melnykovych, or from the American Type Culture Collection (ATCC). Radioimmunoassays and radioreceptor assay. A specific double-antibody radioimmunoassay using hCG and hCG-a as reference standards was used to measure hCG and hCG-a in the culture medium (21). Antisera used were: anti-hCG-f~ (Sb6) for hCG (22), and anti-hCG-a (CA3) for hCG-a {14). Purified preparations of hCG (CRl19; 11,600 IU per rag; ventral prostate weight assay; Second International Standards hCG) and of hCG-a (CR117) were radioiodinated and used as standards and tracers. HCG (CRl19) had 1% to 3% cross-reactivity in the hCG-a as-
775
CHOU
776
TABLE 1 EFFECT OF SODIUMBUTYRATEANDBRDURD ON HCG-t~ ANDHCG SECRETIONa
Strain
Control
hCG-a
hCG
Sodium butyrate
Sodium butyrate
ng / mg protein 82 1 49 124 207 448 917 2,940 2,778 2,450 93 253 560 437 837 550 2,270 926 1,648 13,830
BrdUrd
Control
BrdUrd
ng / mg protein 6.9 114.8 5.9 1,556.0 52.3 69.0 136.7 207.1 303.0 33.3
