Chromosome Analysis and Alkaline Phosphatase of C4I, a Cell … · Chromosome Analysis and Alkaline...
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[CANCER RESEARCH 37, 3209-3213, September 1977] Chromosome Analysis and Alkaline Phosphatase of C4I, a Cell Line of Human Cervical Origin Distinct from HeLa1 Fritz Herz,2 3 Orlando J. Miller,4 Dorothy A. Miller,5 Nelly Auersperg,6 7 and Leopold G. Koss3 Department of Pathology, Montefiore Hospital and Medical Center, Albert Einstein College of Medicine, Bronx, New York 10467 [F. H.,L. G. K.J; Department of Human Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York 10032 [O. J. M., D. A. M.¡;and Cancer Research Centre and Department of Zoology, University of British Columbia, Vancouver, British Columbia, V6T 1W5, Canada [N. A.] SUMMARY The C4I cell line, which was derived from a human squa- mous carcinoma of the uterine cervix, has been character ized by analysis of quinacrine-banded metaphase chromo somes and study of alkaline phosphatase. C4I cells have a distinctive karyotype. They are hypodiploid, with a highly characteristic series of marker chromosomes, most of them derived by translocation or deletion. They contain no HeLa cell marker chromosomes, and the cell line shows no evi dence of HeLa cell contamination. Nevertheless, the C4I and the HeLa cell line, both derived from cervix cancer, although of a different histological type, produce similar alkaline phosphatases. The enzyme is heat stable (placental type), is inhibited by L-phenylalanine, and responds to the inducing effects of prednisolone and/or hyperosmolality. INTRODUCTION The C4I cell line was established by Auersperg and Ha- wryluk (4) in 1961 from an exophytic, Stage II, Grade IV, invasive squamous carcinoma of the cervix of a white woman. This line has retained the ability to form desmo- somes between adjacent cells (3), a feature that is typical of these epithelial cells in vivo. The C4I cells were hypodiploid in the early passages (4) and have B-type glucose-6-phos- phate dehydrogenase (2, 7). The HeLa cell line, which was established by Gey ef al. (10), was derived from a cervical adenocarcinoma (14) of a black woman. Many established human cell lines have been shown to be HeLa contaminants on the basis of having common karyotypic markers (15, 19, 22) and A-type glu- cose-6-phosphate dehydrogenase (9). This report confirms that C4I is not contaminated by HeLa and demonstrates that C4I has a characteristic karyotype with distinctive marker chromosomes different from those seen in HeLa. Nevertheless, both cell lines produce the 1 Supported in part by National Cancer Institute Contract NO1-CB-43963. 2 To whom requests for reprints should be addressed, at Montefiore Hospital and Medical Center, 111 East 210th Street, Bronx, N. Y. 10467. 3 Recipient of National Cancer Institute Contract NO1-CB-43963, during the tenure of which this work was performed. 4 Recipient of NIH Grants GM 22966 and CA 12504, during the tenure of which this work was performed. ' Recipient of support from The National Foundation-March of Dimes, during the tenure of which support this work was performed. • Recipient of support from The National Cancer Institute of Canada, during the tenure of which support this work was performed. 7 Research Associate of the National Cancer Institute of Canada. Received February 24, 1977; accepted May 27, 1977. same heat-stable, inducible form of alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1]. MATERIALS AND METHODS Cell Culture Technique. C4I cells were grown in 75-sq cm plastic flasks using Eagle's basal diploid medium supple mented with 11% fetal calf serum, penicillin (100 units/ml), streptomycin (100 /^ig/ml), and amphotericin B (0.25 /ng/ ml). Medium was changed 3 to 4 times a week. After growth at 37° in a humidified atmosphere of 5% CO.,in air, the cells were transferred as small clumps on a weekly schedule using 0.25% trypsin. Culture of HeLa S3 cells was carried out as described before (13). Where indicated, the osmolal- ity of the culture medium was increased by the addition of appropriate amounts of NaCI (from an autoclaved 3 M stock solution) 24 hr after cell transfer. A stock solution of pred nisolone,8 100 ¿tg/ml,was prepared in ethanol, and appro priate amounts of this solution were added to the cultures 24 hr after cell transfer. Ethanol was added to the controls. In some experiments NaCI and prednisolone were added simultaneously. At the end of the 7-day growth cycle, cells were harvested with trypsin, washed 3 times with cold 0.15 M NaCI, and assayed for enzyme activity after ultrasonic disruption for 2 min with an ice water-cooled Raytheon 10- kc sonic oscillator (13). Chromosome Preparations. Metaphase chromosome preparations were made by a modification of the method described by Miller ef al. (20). Cells were harvested by aspirating with a pipet, treated with hypotonie (0.075 M) KCI, fixed in methanohglacial acetic acid (3:1, by volume), and dropped onto cold, wet microscope slides. After being air dried, the slides were stored at 4°.The cells were stained with quinacrine, and suitable metaphase spreads were photographed on H and W Control film. Chromosomes were counted on photographic prints. Karyotypes were pre pared for 10 cells. In a few cases chromosomes were mea sured with a ruler subdivided 32 units to the inch. Enzyme Assays. Alkaline phosphatase activity was deter mined by the hydrolysis of p-nitrophenyl phosphate (13) using 1 M 2-amino-2-methyl-1-propanol-HCI buffer at pH 10.6 and 38°. Acid phosphatase activity was measured with the same substrate at pH 4.8 using 0.1 M acetate buffer. 8 The trivial name used is: prednisolone, 11/3,17,21-trihydroxypregna-1,4- diene-3,20-dione. SEPTEMBER 1977 3209 on June 10, 2020. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
Transcript of Chromosome Analysis and Alkaline Phosphatase of C4I, a Cell … · Chromosome Analysis and Alkaline...
[CANCER RESEARCH 37, 3209-3213, September 1977]
Chromosome Analysis and Alkaline Phosphatase of C4I, aCell Line of Human Cervical Origin Distinct from HeLa1
Fritz Herz,2 3 Orlando J. Miller,4 Dorothy A. Miller,5 Nelly Auersperg,6 7 and Leopold G. Koss3
Department of Pathology, Montefiore Hospital and Medical Center, Albert Einstein College of Medicine, Bronx, New York 10467 [F. H.,L. G. K.J; Departmentof Human Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York 10032 [O. J. M., D. A. M.¡;and CancerResearch Centre and Department of Zoology, University of British Columbia, Vancouver, British Columbia, V6T 1W5, Canada [N. A.]
SUMMARY
The C4I cell line, which was derived from a human squa-mous carcinoma of the uterine cervix, has been characterized by analysis of quinacrine-banded metaphase chromosomes and study of alkaline phosphatase. C4I cells have adistinctive karyotype. They are hypodiploid, with a highlycharacteristic series of marker chromosomes, most of themderived by translocation or deletion. They contain no HeLacell marker chromosomes, and the cell line shows no evidence of HeLa cell contamination. Nevertheless, the C4Iand the HeLa cell line, both derived from cervix cancer,although of a different histological type, produce similaralkaline phosphatases. The enzyme is heat stable (placentaltype), is inhibited by L-phenylalanine, and responds to theinducing effects of prednisolone and/or hyperosmolality.
INTRODUCTION
The C4I cell line was established by Auersperg and Ha-wryluk (4) in 1961 from an exophytic, Stage II, Grade IV,invasive squamous carcinoma of the cervix of a whitewoman. This line has retained the ability to form desmo-somes between adjacent cells (3), a feature that is typical ofthese epithelial cells in vivo. The C4I cells were hypodiploidin the early passages (4) and have B-type glucose-6-phos-phate dehydrogenase (2, 7).
The HeLa cell line, which was established by Gey ef al.(10), was derived from a cervical adenocarcinoma (14) of ablack woman. Many established human cell lines have beenshown to be HeLa contaminants on the basis of havingcommon karyotypic markers (15, 19, 22) and A-type glu-cose-6-phosphate dehydrogenase (9).
This report confirms that C4I is not contaminated by HeLaand demonstrates that C4I has a characteristic karyotypewith distinctive marker chromosomes different from thoseseen in HeLa. Nevertheless, both cell lines produce the
1 Supported in part by National Cancer Institute Contract NO1-CB-43963.2 To whom requests for reprints should be addressed, at Montefiore
Hospital and Medical Center, 111 East 210th Street, Bronx, N. Y. 10467.3 Recipient of National Cancer Institute Contract NO1-CB-43963, during
the tenure of which this work was performed.4 Recipient of NIH Grants GM 22966 and CA 12504, during the tenure of
which this work was performed.' Recipient of support from The National Foundation-March of Dimes,
during the tenure of which support this work was performed.•Recipient of support from The National Cancer Institute of Canada,
during the tenure of which support this work was performed.7 Research Associate of the National Cancer Institute of Canada.
Received February 24, 1977; accepted May 27, 1977.
same heat-stable, inducible form of alkaline phosphatase[orthophosphoric-monoester phosphohydrolase (alkalineoptimum), EC 3.1.3.1].
MATERIALS AND METHODS
Cell Culture Technique. C4I cells were grown in 75-sq cmplastic flasks using Eagle's basal diploid medium supple
mented with 11% fetal calf serum, penicillin (100 units/ml),streptomycin (100 /^ig/ml), and amphotericin B (0.25 /ng/ml). Medium was changed 3 to 4 times a week. After growthat 37°in a humidified atmosphere of 5% CO., in air, the cells
were transferred as small clumps on a weekly scheduleusing 0.25% trypsin. Culture of HeLa S3 cells was carriedout as described before (13). Where indicated, the osmolal-ity of the culture medium was increased by the addition ofappropriate amounts of NaCI (from an autoclaved 3 M stocksolution) 24 hr after cell transfer. A stock solution of prednisolone,8 100 ¿tg/ml,was prepared in ethanol, and appro
priate amounts of this solution were added to the cultures24 hr after cell transfer. Ethanol was added to the controls.In some experiments NaCI and prednisolone were addedsimultaneously. At the end of the 7-day growth cycle, cellswere harvested with trypsin, washed 3 times with cold 0.15M NaCI, and assayed for enzyme activity after ultrasonicdisruption for 2 min with an ice water-cooled Raytheon 10-kc sonic oscillator (13).
Chromosome Preparations. Metaphase chromosomepreparations were made by a modification of the methoddescribed by Miller ef al. (20). Cells were harvested byaspirating with a pipet, treated with hypotonie (0.075 M)KCI, fixed in methanohglacial acetic acid (3:1, by volume),and dropped onto cold, wet microscope slides. After beingair dried, the slides were stored at 4°.The cells were
stained with quinacrine, and suitable metaphase spreadswere photographed on H and W Control film. Chromosomeswere counted on photographic prints. Karyotypes were prepared for 10 cells. In a few cases chromosomes were measured with a ruler subdivided 32 units to the inch.
Enzyme Assays. Alkaline phosphatase activity was determined by the hydrolysis of p-nitrophenyl phosphate (13)using 1 M 2-amino-2-methyl-1-propanol-HCI buffer at pH10.6 and 38°.Acid phosphatase activity was measured with
the same substrate at pH 4.8 using 0.1 M acetate buffer.
8 The trivial name used is: prednisolone, 11/3,17,21-trihydroxypregna-1,4-diene-3,20-dione.
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Specific activity was expressed as Amólesof p-nitrophenolliberated in 30 min at 38°per mg of protein, the latter
determined according to the method of Lowry ef a/. (16)with crystalline bovine serum albumin as standard.
Thermal Stability and Inhibition Studies. Heat stability ofalkaline phosphatase was investigated by incubating triplicate aliquots of 0.05 ml of cell-free preparations at 56°with 0.1 ml of 1 M 2-amino-2-methyl-1-propanol-HCI buffer(pH 10.6). Controls were incubated with buffer at 4°.After
incubation for various lengths of time, tubes were transferred to 4°,and the remaining activity was subsequentlymeasured at 38°by the addition of 0.1 ml of 0.016 M p-
nitrophenyl phosphate containing 2 mM MgCL. Thermalinactivation studies were also carried out at 56°with 0.1 M2-amino-2-methyl-1-propanol-HCI buffers adjusted to a pHrange between 9.6 and 11.1. The pH of the buffer solutionswas monitored during and after preincubation at 56°.No
significant changes in pH were noted. The proportion ofactivity remaining was computed from controls incubated at4°.Alkaline phosphatase inhibition studies with L-phenylal-anine and L-homoarginine were carried out by including theinhibitor in the complete enzyme assay system (27). For L-phenylalanine, the D-isomer was used as control, and theresults were calculated from the difference in inhibitionbetween the L and the D forms.
RESULTS
Growth Characteristics. The growth characteristics ofC4I are the same as described by Auersperg (3). Twenty-fourhr after transfer, the cell clumps start to attach to the surface, and the cells spread slowly on the growth surface,forming round colonies. When 2 colonies merge, the site offusion is marked by distinct ridges that remain distinguishable for many days. In the stationary phase the culturesbecome stratified, and as seen by the time lapse cinematography, cell division occurs without the cells detaching fromthe growth surface (unpublished observations). Ultrastructural studies indicate that C4I has retained the capacity toform abundant desmosomes between adjacent cells.
Chromosome Analysis. The chromosomes in 41 cellswere counted. There was a mode of 41 to 43 chromosomes/cell; 13 cells had 41, 8 had 42, and 10 had 43 chromosomes.Ten cells, selected on the basis of absence of overlappingand clarity of quinacrine banding pattern, were karyotyped,and a histogram was prepared illustrating the average chromosome complement of a cell (Chart 1). Three-fourths of
the chromosomes had normal banding patterns. There were2 or more normal copies per cell of 10 of the 22 autosomesand the X and single copies of all the other chromosomes,except Nos. 11, 15, and 18. In addition, single copies ofseveral abnormal, or marker, chromosomes were present.Every cell had several marker chromosomes, although notevery marker was present in every cell. The origin of most ofthe marker chromosomes could be determined from theirquinacrine banding patterns (Fig. 1). Mar 1 is a No. 2 chromosome from which the distal one-third of the long arm hasbeen deleted, giving a chromosome with an arm ratio of 1.2compared to 1.6 for the normal No. 2 chromosome. Thelong arm of Mar 2 has the banding pattern of the long arm ofNo. 8, and the short arm has the pattern of the long arm ofNo. 12, but in each case the respective arm is slightly longerin the marker than in the normal copy. Mar 2 is the samelength and has the same arm ratio as the normal No. 3chromosome. The short arm of Mar 3 is probably derivedfrom the long arm of No. 17, and its long arm is probablyderived from the long arm of No. 4. The No. 17 portion isslightly shorter than the long arm of the normal No. 17chromosome; the No. 4 portion is longer than the long armof the normal No. 4 chromosome because it has an additional quinacrine-dull band at the distal end. The arm ratioof Mar 3 is 2.8. Mar 4 is a No. 6 chromosome from which thedistal portion of the long arm has been deleted. Mar 5 lookslike a No. 11 chromosome with an insertion or a duplication of the bright band in the long arm. Mar 6 is a sub-metacentric chromosome, which stains poorly with quinacrine. The long arm may be derived from chromosome 15.Mar 7 is a No. 18 chromosome with additional material atthe distal end. It resembles a D-group chromosome but islonger than any of the normal D-group chromosomes. Mar 8has the banding pattern of the long arm of No. 11. Mar 9 is asmall metacentric chromosome with a single bright band ineach arm. This chromosome may have been derived byduplication of the short arm of No. 11.
Alkaline Phosphatase. The alkaline phosphatase produced by the C4I and HeLa S3 lines tested were very similar.In both, the enzyme had a relatively low specific activity,although the level in the C4I cells was about 5 times that inthe HeLa S3 cells (Table 1). The specific activity of theenzyme was increased severalfold when either cell line wasgrown with prednisolone, 0.5 ¿tg/ml(1.4 p.M) or in hyperos-molar growth medium. The simultaneous presence of bothstimuli resulted in an additive effect on the alkaline phosphatase activity of each cell line (Table 1). As previouslyfound with HeLa S3, the activity of several other enzymes,
I 2 3456 7 8 9 10 11 12 13 14 15 16 17 IS 19 20 21 22 X Y 1 2 345 6 789
Chart 1. Histogram showing mean number of copies per cell of normal and abnormal (marker) chromosomes in 10 karyotyped cells from C4I. Horizontalline, 2 copies/cell.
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Chromosomes and Alkaline Phosphatase of C4I
Table 1
Induction of alkaline phosphatase activity in human cervical cancercell lines
Additions tomedium"None
Ethanol (0.5%)Prednisolone (0.5 /¿g/ml)NaCI (50 rriM)Prednisolone + NaCIEstimated
osmolality(mOsmoles
/kg)284
284284384384Specific
activity'HeLa
S3C4I0.84
4.30.84 3.53.52 33.52.30 15.85.20 42.9
°Prednisolone, ethanol, and/or NaCI were added 24 hr after cell
transfer. Cells were grown as monolayers for 7 days. Alkalinephosphatase activity was determined in duplicate on replicate cultures.
6 ¿¿molesof p-nitrophenol liberated in 30 min at 38°per mg
protein.
Table 2
Thermostability of alkaline phosphatase of human cervical cancercell lines
Triplicate 0.05-ml aliquots of enzyme preparations from HeLa S3and C4I cells were preincubated with 0.1 ml of 1 M 2-amino-2-methyl-1-propanol-HCI buffer (pH 10.6) at 56°for times indicatedand then stored at 4°.Enzyme activity was determined at 38°asindicated in text and related to aliquots kept with buffer at 4°.
% residual enzyme activity
Min at56°None51015202530HeLaS3100.089.677.573.471.365.060.2C4I100.090.078.371.166.163.958.7
including acid phosphatase, was not influenced by the inducing agents (24, 25), nor was the total cell protein or cellmorphology altered.
The similarity of the alkaline phosphatases synthesized byC4I and HeLa S3 cells was also shown by the virtual identityof their thermostability at 56°in the presence of 2-amino-2-methyl-1-propanol-HCI buffer (Table 2). Furthermore, thebase-level and induced alkaline phosphatase activity havethe same thermostability in the C4I cells, just as they wereshown earlier to have in HeLa S3 cells (24). When heatinactivation was performed at 56°with buffers adjusted to
varying hydrogen ion concentrations, the percentage ofresidual enzyme activity was the same with base-level orinduced C4I enzyme preparations at a given pH, irrespectiveof the specific activity (Table 3). Additional evidence for thesimilarity of the enzymes in C41 and HeLa S3, and of thebasal and induced alkaline phosphatase in each, was obtained from inhibition studies. There was 70% inhibition byL-phenylalanine (5 mM) and less than 10% inhibition by L-homoarginine (5 mM) in each case.
DISCUSSION
Cells of the C4I line have a distinctive karyotype. Thereare 41 to 43 chromosomes/cell, with normal copies of allthe chromosomes, except Nos. 11, 15, and 18. There is a
Table 3Effect of pH on thermostability of C4I alkaline phosphatase
Triplicate 0.05-ml aliquots of enzyme preparations were preincubated for 45 min at 56°with 0.1 ml of 0.1 M 2-amino-2-methyl-1-
propanol-HCI buffer (adjusted to pH indicated). No changes in pHwere noted during and after preincubation. Residual enzyme activity was determined by standard assay procedure at pH 10.6 andrelated to aliquots preincubated with buffer at 4°.
% residual enzyme activity
Cells growninBMEBME
+ ethanol(0.5%)BME-t-prednisolone(0.5
/¿g/ml)BME+NaCI(50mM)BME+prednisolone+
NaCIpH9.696.9100.0100.0100.095.8pH
10.192.388.994.699.697.6pH10.683.584.891.481.890.2PH11.177.073.275.077.
A80.9
single copy of No. 2, a finding that was noted earlier (4), and2 copies of No. 3. C4I has no HeLa marker chromosomes,and HeLa has no C4I markers. The only marker that appearsin both lines is a small metacentric chromosome, possiblyan isochromosome. The banding pattern and size of thischromosome resembles that of the short arm of No. 11 inC4I and the short arm of No. 5 in HeLa (8). Taken together,these findings indicate that the C4I and HeLa lines areunrelated and confirm the lack of contamination of the C4Iline by HeLa cells, as shown earlier by the presence ofglucose-6-phosphate dehydrogenase type B only (2, 7).
The problem of contamination of cultures is not restrictedto contamination by HeLa cells. Although HeLa is particularly often involved, a cell culture can be contaminated byany other cell line. Isozymes can be used to check for HeLacontamination (9, 22), but even a battery of 13 enzymes wasinsufficient to distinguish 5 human bladder tumor lines fromeach other; only 3 of them had distinctive enzyme profiles(26). Karyotype analysis provides a particularly sensitivetechnique to ensure against contamination by identifyingmany cell lines uniquely. Nelson-Rees ef al. (23) noted thepresence of distinctive marker chromosomes in culturedcells from 8 different human tumors and could even distinguish between 2 sublines derived from the same tumor. In asimilar study, 2 sublines of mouse L cells were distinguished by their marker chromosomes (1). In the presentstudy, karyotype analysis served to distinguish between celllines with a similar alkaline phosphatase.
The C4I karyotype was studied extensively by Auerspergand Hawryluk (4) when this line was first established, and ithas been possible to correlate the results from this earlierstudy (done without banding) with those of the presentstudy (done with banding) with respect to the A-group chromosomes. In the 10 cells that were karyotyped in the present study, the cells had 5 A-group chromosomes (2 copiesof No. 1,1 copy of No. 2, and 2 copies of No. 3) compared to7 A-group chromosomes reported earlier. However, 2 chromosomes identified as abnormal by banding pattern wouldhave been included as part of the A group in the prebandedkaryotype. Mar 1, which has the entire short arm of No. 2and a deletion of part of the long arm, may be the chromosome Auersperg and Hawryluk (4) described as having ashort arm the same length as that of No. 2 but a centromeric
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F. Herz et al.
index of 1.4. The centromeric index of Mar 1 is 1.2, whilethat of a normal 2 is 1.6. Mar 2 has the same length and armratio as does the normal No. 3 and probably represents the3rd "No. 3" observed by Auersperg and Hawryluk (4). In
general the distribution of the remaining chromosomes isquite comparable in the 2 studies. However, 1 abnormalchromosome, Mar 3, may have been formed since the earlier study, because this chromosome is too long to be fittedeasily into the B group, and no abnormal chromosome ofthis description was observed by Auersperg and Hawryluk(4).
Only a few cell lines have been established from cancersof the uterine cervix. These include, in addition to HeLa andC4I, the ME-180 line (30). When ME-180 was established ithad 48 to 130 chromosomes and was described as "sub-triploid." In a recent study, most of the ME-180 cells had 66
to 68 chromosomes both before and after passage throughX-irradiated mice (6). This line differed from HeLa in itssensitivity to viruses (30), in possessing glucose-6-phos-phate dehydrogenase type B (2, 7), and apparently in lacking HeLa chromosome markers (6).
The alkaline phosphatase produced by C4I cells is similarto that of HeLa S3 cells in its low specific activity, inducibil-ity by prednisolone and/or hyperosmolality, heat stability ofboth the basal and induced enzyme, and extent of inhibitionby L-phenylalanine or L-homoarginine. These are also thecharacteristics of placental alkaline phosphatase or the Regan enzyme seen in the serum of patients with variousforms of cancer (21). Thus, to the extent that the alkalinephosphatase of C4I cells has been characterized, it resembles the carcinoplacental enzyme. Although the mechanisms responsible for the production of placental type alkaline phosphatase in certain human tumors have not beenelucidated, the present results suggest that the gene(s)coding for this isozyme can be expressed in the same manner by C4I and some HeLa cell lines and that this expressionis increased in both by the same inducing stimuli. Theidentification of a similar carcinoplacental enzyme in 2 morphologically and karyotypically dissimilar human cervicalcell lines derived from cancers of different histologicaltypes raises the question of whether this enzyme is produced by all cervical cancers and whether this might havediagnostic significance. However, C4I and HeLa S3 are celllines, and it is unknown whether either of the tumors fromwhich they originated produced the placental type of alkaline phosphatase. Furthermore, not all the HeLa cell linesbehave in the same way. Some HeLa cell lines (e.g., thosedesignated KB, Chang liver, and HeLa S3K) have a muchhigher level of alkaline phosphatase activity (11, 13, 29). Inthe cases of KB (11) and Chang liver (13), this activityconsists of a large heat-labile component and a small heat-stable component. In these lines hyperosmolality and/orglucocorticoids depress the total specific activity, with anincrease in the proportion of the heat-stable and a decreasein the proportion of the heat-labile components (11, 25).The reason for the difference in gene expression in variousHeLa cell lines is unknown, but the existence of such clear-cut differences between related HeLa lines, e.g., HeLa S3Gand HeLa S3K (29), indicates both the need for caution inextrapolating to clinical situations and the usefulness ofcultured cell lines in the study of this problem.
The heat-stable, placental-type alkaline phosphatase isseen in some other cultured human cell lines, e.g., theChaGo and BeWo lines, derived from chorionic gonadotro-pin-producing lung carcinoma (18) and chorionic carcinoma (28), respectively; the HCT-8 line, a carcinoembryonicantigen-producing line derived from an adenocarcinoma ofthe lower intestinal tract (27); and the T24 line, derived froma urinary bladder carcinoma (12), which produces tumor-specific antigen. In T24 cells, glucocorticoids plus hyperos-molar medium produce a synergistic rise in alkaline phosphatase (12) rather than the additive effect seen in HeLa S3and C4I cells. On the other hand, placental alkaline phosphatase is not produced by numerous human lines,e.g., thediploid fibroblastic WI-38 line and its SV40 virus-transformed variant, VA-13, as well as neuroblastoma and Hodg-kin's lymphoma lines (17). Thus, neither cancer per se nor
the conditions of cell culture necessarily provide the stimulus for the expression of placental alkaline phosphatase.What is the stimulus? This question remains unanswered,although the results presented here indicate that more than1 human cervical carcinoma can give rise to cell lines capable of expressing this placental enzyme. A deeper understanding of this process may come from somatic cell genetic studies involving either mutagenesis or cell hybridization (5) to permit genetic analysis of the regulation of theexpression of placental alkaline phosphatase.
ACKNOWLEDGMENTS
We acknowledge the excellent technical assistance of A. Schermer and K.Krishnamurthy.
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Fig. 1. Marker chromosomes from 4 cells of C4I cell line stained with quinacrine. See text for probable origin of these chromosomes.
SEPTEMBER 1977 3213
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1977;37:3209-3213. Cancer Res Fritz Herz, Orlando J. Miller, Dorothy A. Miller, et al. Line of Human Cervical Origin Distinct from HeLaChromosome Analysis and Alkaline Phosphatase of C41, a Cell
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