The proportion of cells labeled with tritiated thymidine as a function of population doubling level in cultures of fetal, adult, mutant, and tumor origin

Printed in Sweden Copyright @ 1976 by Academic Press, Inc. A/I rights of reproduction in any form reserved

Experimental Cell Research 102 (1976) 3 1112

THE

PROPORTION THYMIDINE DOUBLING ADULT,

OF CELLS

LABELED

AS A FUNCTION LEVEL

R. A. VINCENT,

AND JR

TRITIATED

OF POPULATION

IN CULTURES

MUTANT,

WITH

OF FETAL,

TUMOR

ORIGIN

and P. C. HUANG’

Department of Biochemical and Biophysical Sciences, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, MD 21205, USA

SUMMARY The proportion of human fibroblast-like cells in culture able to incorporate tritiated thymidine ([sH]TdR) into their nuclei was shown autoradiographically to decrease with increased population doubling level, but variations were found in the pattern with which this occurred. Cultures of fetal origin (WI38) demonstrated consistently high proportions of labeled cells through 40 population doublings, after which a sharp decrease in the proportion of labeled cells was followed by culture termination. A diploid culture derived from an adult donor exhibited a more gradual decrease in the proportion of labeled cells beginning at low population doubling levels. Six Xeroderma pigmentosum cultures of four complementation groups showed decreases in the proportion of labeled cells intermediate between those of the fetal and adult cultures as expected from their donor ages. Diploid Fanconi’s anemia and progeria cultures from young donors exhibited decreases in the proportion of labeled cells at low population doubling levels, and demonstrated sinusoidal fluctuations in the proportion of labeled cells as a function of population doubling level. A diploid culture derived from a benign tumor in an adult donor differed from the HeLa cell line in that it exhibited an accumulation of unlabeled cells with increased population doubling level, although this occurred at greater population doubling levels than in the fetal culture.

The life span of human diploid cells in culture has been described to consist of three phases: (1) the establishment of a primary culture from biopsy fragments; (2) the period during which cultures may be frequently subcultured; and (3) the terminal phase in which cultures require progressively longer time to reach saturation density, become unable to reach confluency, and are lost [22, 231. The latter phase, associated with the accumulation of cellular debris and cells of altered morphology [7, 22, 431, may be reflective of senescence in 1 To whom inquiries should be addressed. 3-761808

vitro. It is generally agreed that cell cultures derived from normal tissue have a finite life span; the infrequent exception being those cultures which undergo spontaneous transformation [49]. The life span of cell cultures has been shown to be inversely correlated with the age of the donor [22, 351 and the population doubling level of the culture [9,22]. The life span may also be affected by the genotype of the cells. Cultures established from patients with the genetic diseases which in many respects mimic aging (progeria and Werner’s syndrome) exhibit shorter life spans than non-mutant cultures established Exp

Cell

Res

102 (1976)

32

Vincent and Huang

from donors of similar ages [18, 341. These observations suggest that the same deterioration process causing mortality of the organism may also be operative in diploid cultures. Human diploid cultures differ markedly in this respect from malignant human cells, which, by their unlimited growth potential in vitro [17], appear immortal [23]. The senescence of human diploid cultures may involve the gradual accumulation of cells which are blocked at a gap stage of the cell cycle [14] or which require a long time to traverse the cell cycle [31]. These cells would be expected to be unable to incorporate r3H]TdR and to appear in autoradiographs as unlabeled cells. Rapidly proliferating cells, in comparison, would be expected to incorporate r3H]TdR during semi-conservative DNA synthesis. Cristofalo & Sharf [8] indeed showed that following prolonged exposure to [3H]TdR human diploid cultures contained both labeled and unlabeled cells, with the % unlabeled cells proportional to the % culture life span completed. Their observations suggest that the ability to incorporate r3H]TdR may be a measure of the proliferative capacity of a culture. The objective of this study was to determine whether a deficiency in proliferative capacity may be detected in mutant cells by measuring the decrease in the proportion of labeled cells with increased population doubling level. A decreased life span in vitro for cells established from patients with Down’s syndrome has been reported to be due to a decreased rate of cellular replication [46]. In our study, cultures were examined from patients with the genetic diseases Fanconi’s anemia [42], Xeroderma pigmentosum [37] of four genetic complementation groups [25,26], and atypical Hutchinson-Gilford progeria [50]. Spontaneous Exp Cell

Res 102 (1976)

Table 1. Culture characteristics Donor

ATCC’

Patient

Culturea*”

IlO.

llO.d

HG,KH FA,JW XPA, JT XPB, PC XPC,, FL XPC,, GD XPD,, CW XPD*, TK BT, KD HeLa WI38 Non-mutant, PH

1 223 1 199 1 170 1204 1 157 1200

BE12 BE 11 BE 1 BE 10 BE6 BE7

CCL 2 CCL 75 -

age (years)

Donor sex

14 5 7 28 27 16 19 11 26 31 Fetal 42

a A descriotion of the cultures and their sources is given in the Materials and Methods of the text. The abbreviations are HG for atypical Hutchinson-Gilford progeria, FA for Fanconi’s anemia, XP for Xeroderma pigmentosum, and BT for benign tumor. The letters A-D in the XP culture designations represent genetic complementation group assignments [25, 261 with numerical subscripts used to specify more than one culture in a given group. The last two letters of each culture designation are the donor’s initials. * Followine are the total number of metaphases examined and % diploid cells for some cultures: HG, 67 (87%); FA, 86 (90%); BT, 50 (74%); PH, 75 (88%); WI38,48 (78%). Metaphases from donors KH and PH were examined by J. Rary and M. Bender; JW by S. Brefach. Values for the WI38 culture were obtained from the Registry of Animal Cell Lines Certified by the Cell Culture Collection Committee.c From the latter source, the modal chromosome number of the HeLa line was 79, with a range of 39-187. c Designations of the American Type Culture Collection Cell Repository, Rockville, Md 20852. d The XP donors were from the Bethesda (BE) series

WI.

structural chromosome aberrations have been observed in Fanconi’s anemia [15, 16, 24, 30, 471 and Xeroderma pigmentosum [15] cultures, and patients with Fanconi’s anemia [13] and Xeroderma pigmentosum [37] are disposed to malignancy. The biochemical bases of these diseases have been reported to involve enzymatic deficiencies for the repair of DNA crosslinks [45] and thymine dimers [40] in Fanconi’s anemia, a defective incision step [4, SJ and impaired

Labeling

post-replicational repair [27] of thymine dimers in Xeroderma pigmentosum, and the possible deficient repair [ 11, 411 or increased degradation [28, 511 of DNA strand break damage in progeria. Cultures of fetal (WI38), adult, and tumor origin were also examined. The findings of the study suggest that the presence of certain genetic defects or alterations may influence the manner in which the proportion of labeled cells varies with population doubling level.

indices of mutant human cell cultures only in the assay at the lowest population level.

33 doubling

Culture medium Cells were routinely cultured as monolayers on the bottom surface of 250 ml glass culture bottles with 8 ml of medium. The medium consisted of reconstituted Eagle MEM with Earle’s salts (North American Biologicals, Inc., Rockville, Md 20852) fortified with 15 % v/v fetal bovine serum [Grand Island Biological Co. (GIBCO), Grand Island, NY 140721 and 1% v/v antibiotic-antimycotic 100x mixture (GIBCO). The complete medium was filter sterilized before use through 0.22 pm cellulose acetate filters (Millipore Corp., Bedford, Mass 01730).

Primary culture

MATERIALS

AND

METHODS

Origin of cells The Fanconi’s anemia culture was established from a skin biopsy of a patient (JW) who exhibited the classical syndrome. The progeroid culture was derived from a skin biopsy of a patient (KH) who was diagnosed by Dr William Reichel of the Franklin Sauare Ho&al. Baltimore, Md, as a variant of classi& progeria: and presumably the same patient used in other studies [ 11, 28, 411. The progeroid and Fanconi’s anemia cultures were both obtained at passage 1 through Dr Jack Rary of the Johns Hopkins Hospital, Baltimore, MD. The Xeroderma Diwzentosum cultures were established by Dr Jay’Robbins and his colleagues [44], and obtained from Drs Rufus Dav and Wes Dineman of the National Cancer Institute,- Bethesda, Md. The same cultures were also obtained at subcultures 2 to 4 from the American Type Culture Collection, Rockville, Md. Cultures from the latter source were utilized only for the first assay to provide values at low population doublings. Assays at higher population doubling levels were accomplished previously on cells obtained from the NCI. The PH culture was established in this laboratory from a skin biopsy of a normal adult male. The benign tumor culture was derived from a non-invasive, venous ectasia (diagnosis by Dr Peter Howarth of the NIH, Bethesda, Md) on the lip of an otherwise healthy adult female (KD). The tumor appeared on a site where a sham canine tooth had frequently irritated the lip. The tumor culture was obtained from Dr Rufus Day. The HeLa culture, established from a carcinoma of the human cervix [ 171, was obtained as original stock from the late Dr George Gey through Dr Timothy Merz of this institution. All of the cells in this study were libroblast-like, with the exception of the HeLa cells which were epithelial-like. Each of the above cultures and their donors are further described in table 1. The WI38 cultures [22] were kindly made available by Mrs Sandra Harris of the Gerontoloev Research Center at Baltimore City Hospitals and by& Leonard Haytlick of the Stanford Universitv School of Medicine. Stanford, Calif. Cultures from the latter source were used

Biopsy specimens were obtained by pinch biopsy of the skin on the inner aspect of the forearm. The specimens were cut into 1 mm* fragments, 4 to 5 of which were nositioned in 25 cm2 plastic tissue culture flasks (Falcon Plastics, Oxnard, dalif. 93030). The fragments were allowed to become attached before incubation as previously described [21]. When outgrowths covered approx. 75 % of the growth surface, the cells were subcultured. The resulting culture was designated subculture 1.

Subculture procedure The culture medium was routinely changed twice a week. Cells were subcultured as soon as they reached confluency or saturation density. The subculture procedure involved two washes each with 5 ml of pH 7.0 Puck’s balanced salt solution and exposure for 1 to 3 min at 34-37°C to 2 ml of pH 7.1 trypsin solution [33] without antibiotic. Cell suspensions were prepared after removal of the trypsinsolution. The fibroblastlike cultures were generally split 1: 8. The HeLa culture was split 1:45 or 1 :!?O. Population doubling levels for both the fibroblast-like and HeLa cultures were calculated from split ratios with subcultivation into vessels of constant size. The number of population doublings accrued by the HeLa cells when they .were obtained was not known, and the reported values represent the number of doublings accrued in this laboratory since they were obtained: hence, the reported doubling levels are minimum estimates. Fibroblast-like cultures were considered senescent and terminated when low cell densities persisted for l-3 months after the last subcultivation.

Handling cells Cells were cultured at 37.5’C in a humidified atmosphere of 58% CO*. Cultures were exposed only to alass faltered white fluorescent or direct amber fluorescent light. Checks for mycoplasma infection were made by examining autoradiographs for perimembrane incorporation of radiolabel [38] from [3H]TdR (5 &/ml, 20 Ci/mM, 2 h exposure, 7 day film exposure), and by Exp Cell

Res 102 (1976)

34

Vincent and Huang I.

Fig.

1. Abscissa: time of exposure (hours); ordinate: % cells with labeled nuclei. Values beside curves represent subcultivation and ponulation doubling (in brackets) levels. Cells were seeded at 1.0X 103 cells/ cm* on coverslips in 35 mm Petri dishes. After 24 h incubation, medium containing r3H]TdR was added and the cultures were incubated for 24 to 144 h. At

termination cultures were fixed in 3 : 1 ethanol/acetic acid and air-dried following hydration through an alcoho1 series. Each coverslin was then mounted on a slide, autoradiographed, and scored for labeled nuclei. Percent labeled (a) W138; (b) PH (non-mutant) cells as a function of exposure time to [aH]TdR (0.1 &i/ml, 2 Ci/mM).

the direct observation of aceto-orcein stained coverslio cultures [ 121. Although our findings were negative, low levels of infection may escape detection with these methods. All manipulations requiring sterile conditions were carried out in a laminar flow hood biogard, Model B-4000-21.

ScorinQ

Labeling ceils Cultures were assayed according to a modification of the nrocedure of Cristofalo & Shatf T81. Cells were counted with a hemacytometer (A0 In&uments Co., Buffalo, NY 14215) and suspensions were diluted to yield 3 X l(r cells/culture dish (3X 103 cells/cm?. Replicate cultures were prepared in 35 mm plastic Petri dishes, each containing one 22x22 mm glass coverslip. Cultures were incubated for 24 h after seeding, at which time the medium was replaced with 3 ml of fresh medium containine fSHlmethvl thvmidine (0.1 &i/ml, 2.0 Ci/mM). A&r>4 io 144 h, the cells were rinsed in Puck’s balanced salt solution, fixed in two 30 min changes of 3 : 1 ethanol/acetic acid, hydrated serially through 100,95, and 70% ethanol and distilled water, and air-dried. Coverslips were mounted with Eukett (Calibrated Instruments, Inc., Ardsley, NY 10502) on microscope slides with cells in the up position.

Autoradiography Slides with cells on coverslips were coated with film by dipping in undiluted Kodak NTB2 emulsion. The slides were exposed at 4°C for 4 days and processed at 8°C with Kodak Dl9 developer for 5 min, a water rinse for 20 set, and Kodak standard fixer for 5 min. Cell nuclei were stained lightly in Mayer’s hematoxylin for 5 min, blued in running water, air-dried, and made permanent with Eukett. Exp Cell Res 102 (1976)

Cells were examined microscopically to determine the proportion of cells having 20 or more silver grains over each nucleus. The nuclei of most cells were either heavily labeled or had only 0 to 2 silver grains. A minimum of 400 cells were scored for each coverslip. Particular care was taken to examine different regions of the same coverslip, and to score areas where cells were in close proximity but not in direct contact.

RESULTS The proportion of radiolabeled cells in each culture was examined with regard to the kinetics of incorporation of label and its correlation with population doubling level. Figs l-4 show the proportions of labeled cells for each culture following exposure to C3H]TdR for 24-144 h. In most cases, the proportion of labeled cells increased rapidly following the addition of radiolabel until a maximum value was reached. This usually occurred within 72 h exposure to rH]TdR. But some cultures, such as the group A Xeroderma pigmentosum culture at passage 13 (fig. 2), failed to reach a maximum proportion of labeled

Labeling

indices of mutant human cell cultures

35

Fig.

2. Abscissa: time of exposure (hours); ordinate: % cells with labeled nuclei. Values beside curves represent subcultivation and population doubling (in brackets) levels. The experimental protocol is described in the legend to fig. 1.

Percent labeled Xeroderma pigmentosum cells of complementation groups (a) XPA, CRL 1223; (b) XFTI, CRL 1199; (c) XPC,, CRL 1170; (d) XPC,, CRL 1204 as a function of exposure time to [3H]TdR (0.1 &i/ml, 2 CilmM).

cells within the longest exposure time of 144 h. The duration of incubation required to obtain a maximum proportion of labeled cells in some cases increased with population doubling level. Figs l-4 also show that the maximum proportion of labeled cells attainable for a given population doubling level usually decreased with increasing doubling level. This relationship was not consistent in the progeroid, Fanconi’s anemia, and benign tumor cultures. No decrease in the proportion of labeled cells was observed in the HeLa culture . The maximum proportions of labeled cells derived from the plateau segments of the curves in figs l-4 (or the 72 or % h ex-

posure times for those curves which did not level off) were plotted against the respective population doubling levels and shown in figs 5 and 6. From these figures it can first be seen that the control cultures differ in the manner in which labeled cells decrease with increased population doubling level. In this respect, the WI38 culture demonstrated high labeling indices of greater than 95 % for the first 40 population doublings, after which the proportion of labeled cells decreased logarithmically to 5 % in the next 10 doublings. In contrast, the PH culture from the non-mutant donor showed a reduced proportion of labeled cells at low population doubling levels and exhibited a more continuous, gradual decrease over most of the ExpCeNRes

102 (1976)

36

Vincent and Huang

10’33a 90!

10 L. 0 10 20 50 40

50 M

,‘I

80

90 co

I10 ml im 140 IYI

Fig. 3. Abscissa: time of exposure (hours); ordinate: % cells with labeled nuclei. Values beside curves represent subcultivation and population doubling (in brackets) levels. The experimental protocol is described in the caption to fig. 1.

Percent labeled Xeroderma pigmentosum cells of complementation groups (a) XPDr, CRL 1157; (b) XP&, CRL 1200; (c) KD cells derived from a benign tumor; and (d) HeLa cells (cervical carcinoma) as a function of exposure time to [sH]TdR (0.1 &i/ml, 2 Ci/mM).

culture life span of from greater than 70% to less than 10 % labeled cells. The relationship between proportion of labeled cells and population doubling level for each of the Xeroderma pigmentosum cultures was similar to that of the nonmutant PH culture (see fig. 5), but these cells exhibited decreases in the maximum proportions of labeled cells intermediate between those of the PH and WI38 cultures. All of the Xeroderma pigmentosum cultures reached a greater population doubling level than the PH culture, but several of these cultures demonstrated decreased proportions of labeled cells early in their life span. In contrast to these observations, the HeLa culture (also shown in fig. 5) did not show any decreases in the maximum pro-

portion of labeled cells over 420 population doublings. With the exception of the group B culture, the life spans of the Xeroderma pigmentosum cultures from the two sources were similar [table 2, last column]. As shown in fig. 6, the progeroid and Fanconi’s anemia cultures demonstrated downward sinusoidal decreases in the maximum proportions of labeled cells, with initial decreases occurring at relatively low population doubling levels as compared to the non-mutant PH culture. A small fluctuation in the maximum proportion of labeled cells was also observed in the benign tumor culture. This culture did not exhibit significant reductions in the proportion of labeled cells until after 71 population doublings. Table 2 shows the population doubling

Exp Cell Res 102 (1976)

Labeling

indices of mutant human cell cultures

:I, 0

10 20 YI 40

50 M

10

ij;&yz, Y) 10 Yl 60 XI 80 90 ml IO I20 1,o 140Ix)

ml $0 100 110 120 IJO 140 150

Fig. 4. Abscissa: time of exposure (hours); ordinate: % cells with labeled nuclei. Values beside curves represent subcultivation and population doubling (in

levels (obtained from figs 5 and 6) and % life span completed when 50, 37, and 0% (by extrapolation) labeled cells were present in each culture. When the WI38 culture exhibited 50% labeled cells, 91% of its life 100

70-

70.

60 -

60-

37

brackets) levels. Percent labeled (a) progeroid, KH; (b) Fanconi’s anemia cells (JW) as a function of exposure time to [3H]TdR (0.1 &i/ml, 2 Ci/mM).

span has been completed. The Xeroderma tumor and PH cultures had 66-92% of their life span completed at 50% labeled cells. Table 2 additionally shows the progeroid and Fanconi’s

pigmentosum, benign

r

50-

40-

30-

zo-

0

IO

xl

30

40

50

0

,,,,,,,,,,,,,,,, IO

20

Fig. 5. Abscissa: no. of population doublings; ordinate: max. % labeled cells. G-C), XPA, CRL 1223 (Xeroderma pi mentosum cells of corn lementation group A);&-& XpB, CRL 1199;~@?XPC,, CRL 1170; +--+, XP& CRL 1204; O-O, XPD,, CRL 1157; *-*, XPD,, CRL 1200; A-A, non-affected skin, PH; C--O, fetal lung, W138; A-A, cervical car-

30

40

50

60

70

80’

+-+4+-+LeA--126 IIS 1.6 353 uo

cinema, HeLa cells. Cultures were set up as described in the Legend to fig. 1. The plateau values of each curve of figs 14 were used to estimate the % labeled cells at each population doubling level. Maximum % labeled cells as a function of population doubling level. Exp Cell

Res 102 (1976)

38

Vincent and Huang

Fig. 6. Abscissa: no. of population doublings; ordinafe: max. % labeled cells. A-A, Fanconi’s anemia, JW, O-O, progeria, KH; CW, benign tumor, KD; A-A, non-mutant skin, PH; O-0, fetal lung. W138. Cultures were set up as described in fig. 1. The

plateau values of each curve of figs 14 were used to estimate the % labeled cells at each population doubling level. Maximum % labeled cells as a function of population doubling level.

anemia cultures to have reached 50% labeled cells twice, the first time at which only 37 and 55%, respectively, of their life spans were completed. The last time these cultures reached 50% labeled cells, 80 and 94%, respectively, of their life spans were completed. At 37 % labeled cells 68-97 % of the life span was completed for all the fibroblast-like cultures, and the progeroid and Fanconi’s anemia cultures reached 37 % labeled cells only once. The total life span in vitro of the WI38 culture is shown in table 2 to be 53 population doublings, while that of the non-mutant PH culture was 30. With the exception of the HeLa culture which did not exhibit senescence and the benign tumor culture which survived to 93 population doublings,

the life spans of the remaining cultures were between these values. The life spans extrapolated from the curves of figs 5 and 6 were similar to the life spans determined from culture termination (table 2, values in parenthesis).

Exp CeNRes 102 (1976)

DISCUSSION The findings of this study suggest that cultures established from fetal, adult, mutant, and tumor origin may differ markedly in the manner in which the proportion of labeled cells varies with population doubling level. That the proportion of labeled cells usually reached a plateau, as shown in figs 1 to 4, has been discussed by Cristofalo & Sharf [8]. An additional explanation for this ob-

Labeling

Table 2. Population 37 and 0 %”

doubling

level at which

indices of mutant human cell cultures the proportion

of labeled cells reached

39 50,

Population doubling level’ Cultureb

50%

37%

HG, KH FA,JW XPA, JH XPB, PC XPC,, FL XPC,, GD XPDr , CW XPD,, TK BT, KD HeLa WI38 Non-mutant, PH

13,28 [37, 801d 18,31 [55,94] 28 [88] 33 [79] 28 [85] 33 [92] 28 [85] 31 [66] 82 [88] >420 48 [91] 21 [70]

30 [86] 32 [97] 2 ;itj 31 [94] :i Eg’:j 32 [68] 85 [91] >420 :; ::3

0% culture life span 36e (35)’ 37 (33) 29 (32) 37O 42 (42) 24 34 (33) 27 37 (36) 33 34 (33) 34 36 (47) 47 95 (93) >420 50 (53) 47 27 (30)

a Values derived from figs 5 and 6. * The abbreviations are HG for atypical progeria, FA for Fanconi’s anemia, XP for Xeroderma pigmentosum, and BT for benign tumor. c The reported population doubling levels do not include the number of population doublings considered necessary to form the first monolayer from the biopsy fragments. d Numbers in brackets represent % lie span completed as computed by dividing the population doubling level at the given % labeled cells by the population doubling level at culture termination. e Values obtained by extrapolation of the curves in figs 5 and 6. ’ Numbers in parentheses represent population doubling levels at culture termination. @Numbers in the last column represent population doubling levels at culture termination for parallel cultures obtained from different sources (see Materials and Methods) at low population doubling levels.

servation is that intranuclear radiation damage induced by tritium decay may inhibit the further proliferation of cells which incorporated radiolabel [6]. No significant reduction in the amount of radioactivity in the medium was found in cultures exposed to radiolabel for 24 to 144 h, indicating that depletion of radiolabel may not have occurred. Maximum proportions of labeled cell: were obtained from the plateau portions of the curves in figs l-4 in an effort to compensate for variations in the kinetics of cell proliferation inherent to different assays, donors, and doubling levels. This may be significant in light of the observations that the duration of the lag phase of cell growth after subcultivation [32] and the heterogeneity of cell cycle times [ 1, 3 l] are

functions of population doubling levels. It is possible that variation in the length of the cell cycle time was responsible for the more gradual increase in the proportion of labeled cells with increased exposure time to radiolabel evident in some of the curves in figs l-4. The overall reduction in the maximum proportion of labeled cells with increased population doubling level (figs 14) is consistent with the observations of Cristofalo & Sharf [8]. However, decreases in the maximum proportion of WI38 cells occurred after 40 population doublings (fig. 5), rather than earlier as they noted. Possible heterogeneity in mass WI38 cultures may cause this difference [48]. Our results are similar to the variation of total cell counts with Exp Cell Res 102 (1976)

40

Vincent and Huang

population doubling level observed by Hayflick [22] in phase II and phase III. The period during which no change in the maximum proportion of labeled cells occurs with increased population doubling level corresponds to phase II, while the exponential decrease in the proportion of labeled cells prior to culture termination corresponds to phase III. These results suggest that a threshold level of damage is accumulated before the exponential decline, but the nature of this damage is unknown. Our results are also in general agreement with a model which predicts that a logarithmic decline in the proportion of dividing cells should only occur when a significant proportion of cells reach their limiting life span [20]. The logarithmic increase in the percentage of non-dividing cells reported by Merz & Ross [36], however, parallels the observations of Cristofalo & Sharf in that the increase began at low population doubling levels. The differing observations obtained for the WI38 cultures may have been influenced by the inoculum size used in each study. The inoculum used in our assays (3x lo3 cellslcm2) is approximately one-third that used by Cristofalo & Sharf [8]. The growth rate for cells within the range for exponential growth has been shown to be inversely proportional to inoculum size [39]. Cells seeded at higher densities also have a reduced opportunity for proliferation before the onset of density mediated inhibition of thymidine uptake [3]. A minimum inoculum size of 6~ lo2 cells/cm2 has been shown to be required for exponential growth [39]. The inoculum size of 7 cells/cm2 used by Merz & Ross [36] for cloning WI38 cells is at a cell density associated with suboptimal growth conditions [lo] which may have a pronounced effect on cell attachment and the capacity of attached cells to divide. Exp CeNRes

102 (1976)

With increased population doubling level, increased time may be required for cells to recover from damage induced by subculture procedure [29]. Macieira-Coelho et al. [32] have demonstrated decreases, dependent upon population doubling level, in both the fraction of cells synthesizing DNA and the number of cells capable of entering S phase immediately following subculture. In unpublished studies we have observed that routine subculture procedures may be selective against labeled cells in cultures of high, but not low, doubling levels. When PH cultures containing 93% labeled cells (plateau value) were prelabeled for 30 h and then subcultured, the ratio of the % labeled cells in the trypsinized culture to that of a parallel culture exposed only to Puck’s balanced salt solution was unchanged (0.99). But when the same was done for a PH culture containing 28% labeled cells (plateau value), the ratio as given above was markedly reduced (0.49). Unless intranuclear radiation damage from tritium decay causes cells at increased doubling level to be peculiarly susceptible to damage incurred during subculture, selection against labeled (rapidly proliferating) cells at subculture may be a component of senescence in vitro. This is supported by earlier observations that show cultures given high split ratios are capable of more population doublings than those given low split ratios [22] and that fewer population doublings may be exhibited by cells in vitro than in vivo [2]. The nearly identical decline in the maximum proportion of labeled cells observed in the final population doublings as shown in figs 5 and 6 suggests that the number of population doublings a culture may undergo with no change in the maximum proportion of labeled cells significantly affects the life span of the culture. The different number of

Labeling

cell doublings in which a maximum proportion of labeled cells was maintained without change for the adult (pH) and fetal (wI38) cultures is indicative of the importance of donor age on proliferative capacity in vitro [22, 351. It is interesting to note the precipitous decline in the maximum proportion of labeled fetal cells after 40 population doublings, in contrast to the more gradual decline exhibited by the cultures from the older donors. It is also noteworthy that the maximum proportion of labeled cells at the lowest population doubling level for all nontumor cultures was lower than that maintained by the fetal culture for approx. 40 population doublings (figs 5 and 6). The above observations suggest the presence of greater numbers of senescing cells in cultures of adult donors at low population doubling levels, and we might infer that cells of adult origin are more heterogeneous than fetal cells with respect to proliferative capacity. As for cells from patients with genetic defects, our study confirmed Goldstein’s [ 191 earlier observation that the life span of two cultures from sibs afflicted with Xeroderma pigmentosum was not diminished when compared to cultures derived from non-mutant donors. By both radiolabel incorporation (fig. 5) and culture termination (table 2), further credulence was given to the conclusion that the repair defects of Xeroderma pigmentosum cells do not affect cell proliferation. Since in this study all cultures were grown under stringent lighting conditions, the possibility still exists that under constant insult with ultraviolet light impaired proliferation would result. The decreased and inconstant life spans of the progeroid cultures [18, 341 may be a reflection of the fluctuating proportion of cells undergoing proliferative DNA synthesis. Results shown in fig. 6 attest to such

indices of mutant human cell cultures

41

an explanation. We have also noted (results not shown) that these cultures display unpredictable changes in the interval between successive subcultivations. That these fluctuations are due to the suspected repair defects, responses to the presence or absence of nutritional or humoral factors, or recurrent alterations in cell subpopulations [20] remains to be determined. The occurrence of unlabeled cells and eventual termination of the benign tumor culture (figs 3 and 5) indicate that this culture also exhibits senescence in vitro, but at population doubling levels greater than in the fetal culture. In contrast, the HeLa culture (figs 3 and 4) exhibited a nearly complete lack of unlabeled cells with an apparent potential for unlimited proliferation in vitro. These observations suggest the existence of a possible relationship between the process responsible for the formation of unlabeled cells and the capacity for benign or malignant growth. The number of population doublings accrued before each culture reached 50 and 37% labeled cells was measured to determine whether a maximum proportion of labeled cells obtained from one assay could be utilized to estimate the % life span completed. The fluctuating proportions of labeled cells observed for certain cultures (fig. 6) and the somewhat large variation in the % life span completed for the different cultures at 50% labeled cells (Results and table 2) indicate that a single assay may not be sufficient for an accurate prediction of lifespan. However, at 37% labeled cells no fluctuations were evident and a somewhat accurate prediction of culture termination could be made. Simple target theory, incidentally, predicts that at a dose in which 37 % surviving cells are present, each sensitive site has received one hit. Reasonable accuracy for predicting the termination of a Exp Cell Res 102 (1976)

42

Vincent and Huang

culture was also obtained by ex :rapolating the final decreasing segments of the curves in figs 5 and 6 to the axis (see table 2). The authors thank Mrs Patricia Bohdan, Miss Susan Davidovich, and Miss Polly Panitz for their helpful technical assistance. This study was supported by an NIH postdoctoral fellowship (GM57140) to R. A. V. and a grant from the National Foundation March of Dimes (CRBS 300).

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