deoxyribonucleic acid replication in fetal cells
TRANSCRIPT
Sagesaka et at.
the control of gonadotropin and prolactin release. AM J OesrEr GYNEC0I. 1985; 152: 485-93.
17. Lambalk CB, de Koning J, van Kessel 11, van Rees GP, Schoemaker J. Calculation of the intra-assay variation per assay and its relevance to LH pulse detection. IRCS Med Sci 1985; 13: 1183-4.
18. Scheele F, Lambalk CB, Schoemaker J, van Kessel H, de Koning J, van Dieten JAMJ. Patterns of LH and FSH in men during high frequency blood sampling. J Endocrinol 1987; 114: 153-60.
19. Tafurt CA, Sobrevilla LA, DeEstrada R. Effects of proges- tin-estrogen combination and progestational contracep- tives on pituitary gonadotropins, gonadal steroids and sex-hormone binding globulin. Fertil Steril 1980; 33: 261-6.
20. Mais V, Cetel NS, Muse KN, Quigley ME, Reid RL, Yen SSC. Hormonal dynamics during luteal-follicular transi- tion. J Clin Endocrinol Metab 1987; 64: 1109-14.
21. Hall JE, Schoenfeld DA, Martin KA, Crowley WF Jr. Hypothalamic gonadotropin-releasing hormone secretion and follicle-stimulating hormone dynamics during the luteal-follicular transition. J Clin Endocrinol Metab 1992; 74: 600-7.
22. Soules MR, Steiner RA, Clifton DK, Bremner WJ. The effects of inducing a follicular phase gonadotropin secre- tory pattern in normal women during the luteal phase. Fertil Steril 1987; 47: 45-53.
23. Couzinet B, Brailly S, Bouchard P, Schaison G. Progester-
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one stimulates luteinizing hormone secretion by acting directly on the pituitary. J Clin Endocrinol Metab 1992; 74: 374-8.
24. Nillius SJ, Wide L. Variation in LH and FSH response to LH-releasing hormone during the menstrual cycle. J Ob- stet Gynecol Br Commonw 1972; 79: 865-73.
25. Yen SSC, Vandenberg G, Rebar R, Ehara Y. Variation of pituitary responsiveness to synthetic LRF during different phases of the menstrual cycle. J Clin Endocrinol Metab 1972; 35: 931-4.
26. Wildt L, Hutchison JS, Marshall G, Pohl CR, Knobil E. On the site of action of progesterone in the blockade of the estradiol-induced gonadotropin discharge in the rhesus monkey. Endocrinology 1981; 109: 1293-4.
27. Clarke IJ, Cummins JT, Findlay JK. Effects on plasma luteinizing hormone and follicle stimulating hormone of varying the frequency and amplitude of gonadotropin- releasing hormone pulses in ovariectomized ewes with hypothalamo-pituitary disconnection. Neuroendocrinol- ogy 1984; 39: 214-21.
28. Clarke IJ, Cummins JT. GnRH pulse frequency deter- mines LH pulse amplitude by altering the amount of releasable LH in the pituitary glands of ewes. J Reprod Fertil 1985; 73: 425-31.
29. Hanker JP, Nieschlag E, Schneider HPG. Hypothalamic site of progesterone action on gonadotropin release. Horm Metab Res 1985; 17: 679-82.
Deoxyribonucleic acid replication in fetal cells Toshiaki Sagesaka, MD, PhD, *-' Nikolai Boubnov, MD, PhD, Teruaki Okuyama, MD, PhD, Henry Paulus, PhD, ''' and Nilima Sarkar, PhD'' Boston, Massachusetts
OBJECTIVES: Our purpose was to develop a sensitive method for assessing the replication time of specific human genes In cultured fetal cells and for detecting potential replication defects. STUDY DESIGN: Synchronous progression of diploid human fetal lung cells through S phase was achieved by releasing from serum restriction with minimum essential medium alpha modification plus 10% fetal bovine serum, followed by hydroxyurea blockage at the G, /S boundary. Deoxyribonucleic acid replication was studied in permeabilized cells using mercurated nucleotides to label nascent deoxyribonucleic acid. RESULTS: A high degree of synchrony in traversal of S phase was Indicated by flow cytometry and a well-defined 7-hour period of deoxyribonucleic acid synthesis. The replication of the topoisomerase 11 gene occurred in a narrow time span 3 hours after entry into S phase. CONCLUSIONS: Fetal cells have been highly synchronized at the beginning of S phase, and the replication time of a specific gene can be defined within a narrow time window. (AM J OasTEr GVNECO4. 1994; 170: 468-73. )
Key words: Cell cycle synchronization, S phase, human fetal fibroblasts, chromosome replication, topoisomerase 11
From the Department of Metabolic Regulation, Boston Biomedical Research Institute, ' and the Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School. ' Supported by United States Public Health Service grant No. GM25022 from the National Institute of General Medical Sciences (N. S. ) and Research Fellowship No. 13-408-901 from the American Heart Association, Massachusetts Affiliate (N. B. ).
Received for publication April 26,1993; revised September 8,1993; accepted September 29,1993. Reprint requests: Toshiaki Sagesaka, MD, PhD, Department of Obstetrics and Gynecology Teikyo University School of Medicine, Ichihara Hospital, Chiba, 299-01, Japan. Copyright C 1994 by Mosby-Year Book, Inc. 0002-9378/94 $3.00 +0 6/1/51815
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Volume 170, Number 2 Am J Obstet Gynecol
Rapid fetal growth in a term depends on rapid cell division in each organ. However, no reports have been
published about the temporal order of normal human diploid deoxyribonucleic acid (DNA) replication, which is an essential feature of cell division, although we reported such studies on heteroploid animal cells. ' To
provide a starting point for investigation of the factors
that influence cell division during fetal tissue growth, we
establish conditions under which normal human diploid
cells proceed through the S phase of the cell cycle in a highly synchronous manner and a sensitive method for
measuring the replication of specific genes during pro-
gression through the S phase. All of the studies on chromosome replication in
mammalian cells to date have been carried out with heteroploid cell lines (i. e., cells with undefined and variable chromosome numbers). Because heteroploidy
may result from defective control of chromosome rep- lication, it is of interest to extend these studies to a normal human diploid cell line, in which the mecha- nisms that control chromosome replication may be
more finely tuned.
Material and methods Materials. IMR90 and IMR91 were obtained from
the Institute for Medical Research in Camden, New
Jersey. IMR90 is a female and IMR91 a male normal human diploid cell line. Both of them originated from
fetal lung cells. Dulbecco's modified essential medium,
minimum essential medium alpha modification, fetal
bovine serum, calf serum, and trypsin were obtained from Gibco. Penicillin-streptomycin, hydroxyurea, ribo-
nuclease type IIIA, and 5-meruri-deoxycytidine tri-
phosphate were obtained from Sigma. Propidium io-
dide was from Calbiochem. Hybridization probe for
human topoisomerase II was from Dr. James C.
Wang. Thiol agarose (Affi-Gel 401) was obtained from
Bio-Rad. Cell culture. The cells were maintained as monolay-
ers in Dulbecco's modified essential medium-10% fetal
bovine serum-l% penicillin-streptomycin (final concen- tration 100 U of penicillin and 100 µg streptomycin per
milliliter). IMR90 was plated out at the population doubling level 24.5 (10 x 105 cells per 75 cm2) for
growth in Dulbecco's modified essential medium and at 25.3 (8 x 105 cells per 75 cm2) for growth in minimum
essential medium alpha modification. IMR91 was plated out at the population doubling level 26.4
(9.2 x 105 cells per 75 cm`) for Dulbecco's modified
essential medium and 26.1 (8 x 10' cells per 75 cm2) for minimum essential medium alpha modification.
Cell synchronization. The detailed method for syn- chronization by serum restriction and hydroxyurea is as follows. 2 Cells were plated out in Dulbecco's modified
Sagesaka et al. 469
essential medium-10% fetal bovine serum-1% penicil- lin-streptomycin into 75 cm' flasks at the density men- tioned above. After 6 hours the medium was removed and the cells were divided into two groups. Group I was maintained in Dulbecco's modified essential medium- 10% fetal bovine serum-1% penicillin-streptomycin, and group 2 was exposed to minimum essential me- dium alpha modification-10% fetal bovine serum-1% penicillin-streptomycin. After 18 hours the cells of groups I and 2, respectively, were washed twice with unsupplemented Dulbecco's modified essential me- dium or minimum essential medium alpha modification and then exposed to Dulbecco's modified essential medium-0.1% fetal bovine serum or minimum essential medium alpha modification-0.1% fetal bovine serum. No antibiotics were used at this or later stages of culture. After 72-hour serum restriction the serum- deficient medium was replaced either with Dulbecco's
modified essential medium-10% fetal bovine serum or minimum essential medium alpha modification-10% fetal bovine serum to release the cells from Go. Nine hours later hydroxyurea was added to each flask to a final concentration of 0.5 mmol/L to arrest the cells at the GI/S boundary. After the cells had been blocked
with hydroxyurea for 9 hours the medium was removed, the flasks were washed with Dulbecco's modified essen- tial medium or minimum essential medium alpha mod- ification, respectively, and new Dulbecco's modified es- sential medium-10% fetal bovine serum or minimum essential medium alpha modification-10% fetal bovine serum was added to release the cells from the G, /S boundary. Four or five 75 cm' flasks were used for each point.
Harvesting cells. At hourly intervals the cells were harvested and processed as follows. After the medium was removed by aspiration, the cells were washed with calcium-magnesium-free phosphate-buffered saline so- lution. The cells were harvested in calcium-magne- sium-free phosphate-buffered saline solution with trypsin, and 10% calf serum was added to terminate trypsinization. Calf serum was removed immediately, and the number of cells was counted with a Coulter counter. A volume that contained 3x 10" cells was processed for DNA synthesis and 1x 10" cells for flow cytometry.
Processing of flow cytometry. The cells were sus- pended in calcium-magnesium-free phosphate-buff- ered saline solution-0.05% Triton X-100 after twice washing with calcium-magnesium-free phosphate-buff- ered saline solution. Ribonuclease type II1A (200 U/ml)
was added, and the cell suspension was incubated for 30
minutes at 37° C and then washed twice. Propidium iodide was added to a final concentration of 50 mg/ml, and the cell suspension was left at 4° C for 1 hour or
470 Sagesaka et at.
overnight. The cells were filtered through 44 nm nylon mesh before flow cytometry with a Coulter model Epics 752 instrument.
DNA synthesis in permeabilized cells. The cells were permeabilized at different stages of S phase by treatment with lysolecithin (50 mg/ml) for 1 minute, followed by DNA synthesis in the presence of mercury- labeled deoxycytidine triphosphate. s Permeabilization was monitored by trypan blue uptake. To study the time of replication of specific genes, we exploited the fact that permeabilized cells can use mercury 5-labeled deoxycytidine triphosphate as a substrate for DNA replication nearly as effectively as normal deoxycytidine triphosphate. '' ' The mercurated nucleotide was incor-
porated into the newly replicated DNA strand, which was monitored with tritiated deoxythymidine triphos- phate. The optimal incorporation was achieved during a 20-minute incubation (10 pmol/10° cells).
Isolation of nascent DNA. Nascent mercurated DNA
was isolated by a slight modification of the procedure of Taljanidisz et al. ' Permeabilized cells (3 x 106) labeled
with mercurated deoxycytidine were lysed by suspen- sion in 0.25 ml of 25 mmol/L ethylenediaminetetra- acetic acid plus 0.5% sodium dodecyl sulfate. The DNA in the cell lysate was sheared by ultrasonic treatment to an average size of 1 to 2 kb and treated with protein kinase K (200 µg/ml) for 1 hour at 37° C, followed by
phenol treatment and concentration by 2-butanol and then ether extraction. After gel filtration on Bio-Gel P4 in saline-sodium citrate buffer to separate DNA from
unincorporated 'Hg-labeled deoxycytidine triphos- phate, the pooled DNA fractions were supplemented with formamide to a final concentration of 60% and heated at 65° C for 5 minutes. Mercurated DNA was purified by two cycles of affinity chromatography' on a column (0.5 ml) of thiol agarose (Affe-Gel 401), equili- brated with a buffer containing 0.1 mol/L sodium chlo- ride, 50 mmol/L Tris-hydrochloric acid, pH 7.5,1 mmol/L ethylenediaminetetraacetic acid, and 60% formamide, using successive washes with 60% forma-
mide (5 ml), 0.1 mol/L sodium chloride (15 ml), water (1.5 ml), 2 mol/L sodium chloride (3 ml), and 0.1 mol/L sodium chloride (6 ml), followed by elution with 0.2 mol/L 2-mercaptoethanol (6 ml). The mercurated DNA in the final eluate was freed of mercaptoethanol by extraction with water-saturated ether and concentrated in a centrifugal vacuum evaporator (Speed-Vac, Sa- vant). By this procedure 85% to 95% of the mercurated DNA applied to thiol agarose was recovered.
Hybridization of nascent DNA. The affinity-purified mercurated DNA was labeled with phosphorus 32 to high specific activity (10" to 10° counts/min/µg) by incubation with the Klenow fragment of DNA poly- merase I, random hexanucleotide primers (14 A2,,, /ml), [a-'$P]deoxyadenosine triphosphate (3000 Ci/mmol),
February 1994 Am J Obstet Gynecol
and the other unlabeled deoxynucleoside triphos- phates, followed by alcohol precipitation in the pres- ence of 2 mol/L ammonium acetate, essentially as de-
scribed by Taljanidisz et al. ' Dot blots of denaturated DNA derived from plasmid pBS-hTOP2, which carries the gene encoding human topoisomerase II (a gift from Dr. James C. Wang, Harvard University) were prepared on 0.54 µm Nyron membranes (Micron Separations No. 10836) with 1µg DNA per dot. The dot blots were washed for 1 hour at 55° C with 2x saline-sodium citrate buffer plus 1% sodium dodecyl sulfate to remove loosely bound DNA and then prehybridized for 1 hour
at 37° C with a buffer containing 50% deionized forma-
mide, 0.75 mol/L sodium chloride, 50 mmol/L sodium phosphate, pH 7.4,5 mmol/L ethylenediaminetetra- acetic acid, 5x Denhardt's solution, 1% sodium do- decyl sulfate, and 0.2 mg/ml denatured salmon sperm DNA. Hybridization with 92P-labeled DNA (2 x 106
counts/min/ml) was performed in the same buffer for 24 hours at 37° C, followed by washing as described
earlier. '
Results
Cell synchronization. After many different condi- tions for cell synchronization were explored, it was found that the simple expedient of changing from Dulbecco's modified essential medium to minimum essential medium alpha modification, followed by arrest at the G1/S boundary by hydroxyurea blockade and subsequent release, afforded excellent cell cycle pro- gression of human diploid IMR90 (Fig. 1) and IMR91 (Fig. 2) cells. By means of flow cytometry cells synchro- nized in Dulbecco's modified essential medium showed broad peaks throughout S phase, especially at 6 hours,
and replication seems incomplete at 7 hours compared with the cells synchronized in minimum essential me- dium alpha modification. The cells grown in the two media were at nearly the same population doubling level (24.5 and 25.3 for IMR90 cells and 26.4 and 26.1 for IMR91 cells), and the observed difference can there- fore not be ascribed to differences in cell age.
Separation of unmodified and mercury-labeled DNA by chromatography on thiol-agarose. On chro- matography on small columns of thiol agarose almost 100% of unmodified DNA was eluted without being bound, whereas about 80% of mercury-labeled DNA was bound and could be eluted with mercaptoethanol (Table I). The mercury-labeled DNA fraction was thus not only recovered in good yield but entirely free of nonmercurated DNA.
DNA synthesis in permeabilized IMR90 cells. The
cells were pulse labeled with mercury-labeled deoxycy- tidine triphosphate every hour during their progression through S phase to yield 10 mercury-labeled deoxycy- tidine triphosphate fractions, each representing the
Volume 170, Number 2 Ani J Obstet Gynecol
DMEM PDL246)
MEM-alpha (PVL25 3,
Fla MR -0 to Ghnq to Allen Add hv*u Rw. .
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a we. e Nwe mow
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Sagesaka et al. 471
Fig. 1. Effects of culture medium on synchronization of 1MR90 cells. Female human fetal lung fibroblasts (IMR90 at population doubling level of about 25) were subjected to cell synchronization protocol described in Material and methods and outlined in figure, with either I)ulbecco's modified essential medium (DMEM) or minimurn essential medium alpha modification (MEA1-alpha) as indicated. Samples were taken at hourly intervals after entry into S phase and subjected to flow cytometric analysis as described in Material and methods. Results of' flow cytometric analysis are shown above time scale, and diagrammatic representation of corresponding cell-cycle phase is shown below.
DMEM PD128 4)
MEM-alpha (PDL26 1)
0 5 za Puuan CWV m Chop to
mwwaw 10%wm tl%+
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Fig. 2. Eflect of culture medium on synchronization of Mw ii I1,. ýI, il Iniin, n fetal lung Ithroblasts (IMR91 at population doubling level of' about 26) were suhjecteel to outlined cell synchronization protocol with either Dulbecco's modified essential medium (UMEM) or minimum essential medium alpha modification (MEM-alpha) and results presented as described in Fig. 1.
1)NA replicated within a narrow span of each point. Replication time of human topoisomerase II. From Comparison of the amount of radioactivity incorpo- the relative hybridization of gene-specific probes with rated into each traction slowed that S phase was cow- the mercury-labeled t)NA labeled at various times dur- pleted in 7 hours (Fig. 3). ing S phase it is possible to deduce the time in S phase
GI/S(Oh) (Ih) (S") (3h) (M) (Sh) (6h) (7h) (10ß)
G1IS(Oh) (1h) (M) (3h) (4h) (Sh) (6h) (1h) Ah)
472 Sagesaka at al.
V1
U U e 0 s d ö E a
Fig. 3. Incorporation of tritiated deoxythymidine triphos- phate into DNA in permeable IMR90 cells at various times during traversal of S phase. Samples of cultured IMR90 cells synchronized at the G1/S boundary were removed at hourly intervals after release from hydroxyurea blockage, as described in Fig. 1. Cells were permeabilized by treatment with lysolec- ithin, and incorporation of tritiated deoxythymidine triphos- phate into DNA was measured by methods of Taljanidisz et al. ' as outlined in Material and methods.
c rn la c 0
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February 1994 Am J Obstet Gynecol
Fig. 4. Replication time of the topoisomerase II gene during S phase in IMR90 cells. Permeabilized samples of synchro- nized IMR90 cells, described in Fig. S, were labeled with 'Hg deoxycytidine triphosphate, and mercurated DNA was isolated by affinity chromatography on thiol-agarose and analyzed for human topoisomerase Il-specific sequence by methods of Taljanidisz et al., ' which involved hybridization to immobilized gene-specific DNA as outlined in Material and methods.
Table I. Purification of mercury-labeled-DNA by chromatography on thiol-agarose
Radioactivity eluted from column
Fraction Loaded with control DNA
(counts/min) Loaded with mercurated DNA
(counts/min)
Unbound 550,000 33,900 Sodium chloride wash (0.1 mol/L) 540 1,650 Water wash 0 150 Sodium chloride wash (2 mol/L) 60 670 Sodium chloride wash (0.1 mol/L)* 1,440 14,180 Mercaptoethanol eluate (0.2 mol/L) 30 168,000
DNA synthesis was allowed to proceed for 1 hour at 37° C in permeabilized human cells in the presence of [a-"'PI deoxyadenosine triphosphate, without or with mercury-labeled deoxycytidine triphosphate, and the DNA was isolated, separated from unincorpo- rated nucleotides by chromatography on BioGel P4, and then fractionated at 25° Con columns of thiol-agarose (BioRad Affigel 401). DNA samples (about 500,000 counts/min of control DNA and 200,000 counts/min of mercurated DNA) were applied to columns in a buffer containing 60% formamide. Columns were washed and eluted as described in Material and methods.
*After overnight storage at 25° C.
at which each of these genes was replicated. As illus- trated in Fig. 4, the most active time of replication of human topoisomerase II gene was at 3 hours, just in the middle of S phase, corresponding to aC value of about 2.9 (DNA content of about 2.9 haploid equiva- lents).
Comment We could achieve highly synchronous passage of
IMR90 and IMR91 cells through S phase by release from serum limitation in the presence of minimum essential medium alpha modification, followed by hy- droxyurea blockade at the G1/S boundary. When Dul-
becco's modified essential medium was used in place of minimum essential medium alpha modification, cell cycle synchronization was much less effective. Flow
cytometry of cells synchronized in Dulbecco's modified essential medium showed broad peaks through S phase, and the replication was incomplete at 7 hours com- pared with cells synchronized in minimum essential medium alpha modification (Figs. 1 and 2). Tobey et al. 7 reported earlier that human foreskin fibroblasts
could be more effectively synchronized at the Go/G1 boundary by using minimum essential medium alpha modification rather than Dulbecco's modified essential medium during release from serum limitation.
Time after entry into S phase (hours) Time after entry into S phase (hours)
Volume 170, Number 2 Ani) Obstet Gynecol
The reason for the superior synchrony in minimum
essential medium alpha modification compared with Dulbecco's modified essential medium is not clear, but
it is possible that growth medium composition affects
paracrine secretions by cultured cells, which may favor
synchronous traversal of S phase. There have been a
number of reports that specific growth factors contrib-
ute to fetal growth. Hill et al. "- " reported that human
placental lactogen indirectly stimulates DNA synthesis in human fetal connective tissue through the paracrine
release of insulin-like growth factor-I-somatomedin-C. Strain et al. '° reported that insulin-like growth factors
play an important role in paracrine regulation of hu-
man fetal growth. Bertrand et al. " showed that epider-
mal growth factor can directly influence human fetal
kidney, supporting the hypothesis of Adamson and Meek" that epidermal growth factor initially stimulates
proliferation in fetal tissues and then stimulates differ-
entiation as the tissues mature. Nagasaka et al. " re-
ported that basic fibroblast growth factor, the active
mitogen and angiogenic factor, participates in the for-
mation of the human placenta. The methods described
in this paper should allow the determination of whether
any of these factors indeed influences the progression through S phase.
The achievement of a high degree of synchrony in
passage through S phase paves the way for analysis of
the temporal pattern of gene replication. To demon-
strate the feasibility of such analysis, we examined the
replication of the topoisomerase II gene using the
mercurated nucleotide technique developed in our lab-
oratory. " The results in Fig. 4 show that this gene, located on chromosome 17, replicates at 3 hours after
entry into S phase at aC value of 2.9. The sharp
transition from the nonreplicating to the replication
state of the topoisomerase 11 gene is an indication of
the synchronous traversal of the cell population through S phase and of the temporal resolution of the
method for analyzing the replication of specific genes. There are some other growth factors that contribute to fetal growth. However, it is unknown where and when
these growth factors affect in the cell cycle. In conclusion, we have developed an effective syn-
chronization procedure for human fibroblasts and a
sensitive method for measuring the replication of spe-
cific genes during progression through S phase. It
Sagesaka at al. 473
should be possible to apply these methods to the determination of the replication time of genes and to investigate how those growth factors stimulate in the cell cycle. This will open the way for prenatal screening for abnormalities in which the DNA replication pro- gram of the fetus may be disturbed.
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