Newly Established Uterine Cervical Cancer Cell Line …cancerres.aacrjournals.org/content/canres/43/4/1748.full.pdfCancer Research Center, La Jolla Cancer Research Foundation, La Jote,

[CANCER RESEARCH 43, 1748-1760, April 1983]0008-5472/83/0043S02.00

Newly Established Uterine Cervical Cancer Cell Line (SKG-III) with

Regan Isoenzyme, Human Chorionic Gonadotropin /?-Subunit, andPregnancy-specific ÃŸi-Glycoprotein Phenotypes1

Shiro Nozawa,2 Yasuhiro Udagawa, Hiroaki Ohta, Soju Kurihara, and William H. Fishman

Cancer Research Center, La Jolla Cancer Research Foundation, La Jote, California 92037 [S. N., W. H, F.], and Department of Obstetrics and Gynecology, School ofMedicine, Keio University, 35 Shinanomachi, Shinjukuku, Tokyo 160, Japan [S. N., Y. U., H. O., S. K.Â¡

ABSTRACT

The production of Regan isoenzyme (heat-stable, t-phenylal-anine-sensitive term-placental alkaline phosphatase). humanchorionic gonadotropin 0-subunit, and pregnancy-specific ÃŸ,-

glycoprotein by newly characterized human uterine cervical cancer cell lines, SKG-llla and SKG-lllb, is reported. These cell lines

were derived from a moderately differentiated epidermoid cancerpartially mixed with epidermoid clear-cell components. At the

end of the first 4 months in culture 2 sublines with differentmorphologies were identified. In nude mice, SKG-llla produceclear-cell epidermoid cancer with much glycogen, while SKG-lllb

grew as a moderately differentiated epidermoid cancer rich intonofilaments. The presence of Regan isoenzyme was established by biochemistry, enzyme cytochemistry, immunocyto-chemistry, and immunoelectrophoresis. However, the copres-

ence of small amounts of early placental alkaline phosphatasewas also demonstrated. The alkaline phosphatase specific activities of SKG-llla cells and SKG-lllb cells were 3.7 and 1.4 nmol

per mg protein per min, respectively. The existence was provenby radioimmunoassay of human chorionic gonadotropin 0-sub-unit (SKG-llla, 5.0 mlU/mg protein; SKG-lllb, 4.4 mlU/mg protein),pregnancy-specific /Vglycoprotein (SKG-llla, 0.7 ng/mg protein)

in the culture media as a tumor cell product. The described celllines may serve as a more representative model system forstudies of regulation of oncodevelopmental genes in gynecological tumors in general and in epidermoid cervical cancer inparticular.

INTRODUCTION

Together with oncodevelopmental proteins such as a-fetopro-tein, carcinoembryonic antigen, and SPi,3 term-placental ALP

[Regan isoenzyme (11, 24, 38)] has been attracting attention asone of the markers for abnormalities in gene expression associated with neoplasia. Regan isoenzyme, a heat-stable and L-phenylalanine-sensitive ALP, was first believed to exist only in

the trophoblast of the term placenta or in certain types ofcancers. Later, however, Nozawa ef al. (28, 29), in a histochem-ical investigation, reported that the Regan-like isoenzyme was

present in uterine cervical reserve cells which are thought to bethe precursors of most uterine cervical cancers. Further, Nozawa

'This work was supported in part by Grants R01-CA-21967, P01-CA-28896,

and P30 CA30199-01 from the National Cancer Institute, NIH, Bethesda, Md.2To whom requests for reprints should be addressed.3The abbreviations used are: SP,, pregnancy-specific /J,-glycoprotein; ALP,

alkaline phosphatase; HCG, human chorionic gonadotropin; /i-HCG, human chorionic gonadotropin, Â¿-subunit;PAS, periodic acid-Schiff; G-6-PD, glucose-6-phos-phate dehydrogenase; HPL, human placental lactogen.

Received March 13, 1982; accepted January 10, 1983.

ef al. (30) reported that the ALP isoenzyme of reserve cellsunderwent a change, "enzyme deviation," in the course of

uterine carcinogenesis. These facts suggest a possible relevanceto the clinical pathology of uterine precancerous lesions, but atthe same time they require further research in order to understand the biological meaning of enzyme deviation.

Cultured HeLa cells (14) have been utilized for many years asputative epidermoid cervical cancer, but the origin of the HeLacells was questioned by Jones ef al. (18), who suggested theirderivation from adenocarcinoma with obvious glandular elementsrather than from an epidermoid cancer. Moreover, over the years,a great many HeLa sublines have been cultured which exhibitmuch phenotypic heterogeneity. Today one would hesitate tointerpret the behavior of these cells as typical of cervical epidermoid carcinoma. Therefore, the establishment of a cell line derived unequivocally from epidermoid cancer of the uterine cervixis needed.

Some previous workers (2,12,17, 26, 33) have reported suchlines, and recently Pattillo ef al. (32) and Nozawa ef al. (31) haverespectively introduced the DoT cell line with HCG and the SKG-I line with heat-stable, L-phenylalanine-sensitive ALP isoenzyme.

The investigation of oncodevelopmental gene expressionwould be assisted greatly if such new cell lines expressed aspectrum of embryonic gene products. Recently, we have succeeded in establishing such a new uterine cervical epidermoidcancer cell line (SKG-III) which expresses such products asRegan isoenzyme, 0-HCG, and SPt. In this paper, we describe

the process of its establishment and its histological characteristics, together with the characterization of ALP of cultured cells.

MATERIALS AND METHODS

Cell Culture

Culture Material. Uterine tumor tissue for culture was obtained asep-tically on July 26, 1978, by radical hysterectomy of a 38-year-old Japa

nese patient with Stage II uterine cervical cancer who visited KeioUniversity Hospital, Tokyo, Japan. Histologically, moderately differentiated dark epidermoid cancer cells occupied most of the tumor; however,minor components of "clear cells" with abundant, optically clear cyto

plasm and uniform hyperchromatic nuclei were also present. PAS stainwas positive in the clear cells but was negative in the dark cells (Fig. 1).The original tumor from which SKG-III was established was diagnosed

as a moderately differentiated epidermoid cancer partially mixed withepidermoid clear cell components.

Culture Method. The minced tumor was stirred for 30 min at roomtemperature in a 0.25% trypsin solution (Nutritional Biochemical Corp.,Cleveland, Ohio) and centrifugea at 900 rpm (150 x g) for 10 min. Thesediment was suspended in Ham's F-12 medium containing 20% new

born calf serum, 100 units of penicillin, and 100 jug of streptomycin perml at 37Â°under 5% CO2. The studies of growth curve, plating efficiency,

1748 CANCER RESEARCH VOL. 43

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Uterine Cancer Cells with Regan ALP, ÃŸ-HCG,and SP,

chromosome analysis and heterotransplantation, and electron microscopic observation of cultured cells were carried out by the methodsalready published (16).

Electrophoresis of G-6-PD. On Cellogel membrane, horizontal elec-

trophoresis was carried out in 0.06 M barbital buffer, pH 8.6. The stainfor the detection of the enzyme was carried out by the methods ofRattazzi ef al. (34).

ALP of Cultured Cells

Preparation of Extracts. Cells were harvested at confluency byscraping them off with a rubber policeman and washing them with 0.05M Tris-HCI buffer, pH 8.6.

Enzyme extraction was performed by stirring with 50% (v/v) 1-butanol

at room temperature for 1 to 2 hr. The enzyme solution was dialyzedagainst 0.01 M Tris-HCI buffer, pH 8.3, with 1 ITIMMgCI2 at 4Â°overnight.

Enzyme Activity Assay. Total ALP activity of cell extracts wasdetermined by the release of p-nitrophenol from p-nitrophenyl phosphateat 37Â°in 30 min. Inhibition studies were performed by adding 25 n\ of

various concentrations of such organ-specific inhibitors as L-phenylala-nine, L-levamisole, or L-leucine to 25 n\ enzyme solution, and the activities

remaining were measured. Heat stability of the enzyme was determinedby assaying activity remaining after heating of samples for 5 or 15 minat 65Â°.The activities of treated samples were compared with those of

corresponding nonheated controls and were expressed as percentages.Protein assay of cultured cells was carried out by the method of Lowryer al. (20).

Gel Electrophoresis. SKG Illa cells were grown to confluency in RPMImedium, supplemented with antibiotics and 10% fetal calf serum. Theywere washed twice with 50 mM Tris:0.85% NaCI solution, pH 7.5, andscraped from the plates. The cell pellet was resuspended in 2.5 ml ofcold 50 mwi Tris:0.85% NaCI solution, and an equal volume of coldbutanol was added. After the cell suspension was stirred in the cold for1 hr, it was spun at 1000 rpm for 30 min, and the aqueous phase wascollected (enzyme). Electrophoresis was carried out in a Triton gel (10%acrylamide:0.4% Triton) according to the method of Fishman (9), but thestaining was performed in a 1:1 mixture of Naphthol AS-MX phosphate

(20 mg in 100 ml 0.2 M Tris, pH 8.9) and Fast BB blue salt (120 mg in100ml distilled water).

Enzyme Cytochemistry by Light Microscopy. Cultured cells werefixed for 10 min in Baker's 10% formol calcium (3) and washed for 30

min in 0.2 M Tris-HCI buffer, pH 8.9. Cells were preincubated separatelyin the buffer containing L-phenylalanine (40 mw) or L-levamisole (2 mw)

for 30 min at room temperature and then transferred to amino acid (20mM L-phenylalanine: 1 HIM L-levamisole)-containing mixtures. The incubation medium consisted of 5 mg of Naphthol AS-MX phosphate (Sigma

Chemical Co., St. Louis, Mo.) as the substrate and 30 mg of fast violetB salt (Sigma) in 50 ml of 0.1 M Tris-HCI buffer, pH 8.9. Incubation timewas 30 to 45 min at room temperature. For the control of L-phenylalanineinhibition, the incubation medium contained o-phenylalanine, which is anoninhibitory stereoisomer of L-phenylalanine.

Simultaneously, staining was performed with or without heat inacti-vation at 65Â°for 30 min in buffer to evaluate the presence of heat-stable

ALP.Electron Microscopic Enzyme Cytochemistry. SKG-III cells were

fixed in situ in 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, onice for 30 min, washed in 0.05 M cacodylate buffer for 1 hr, and thenincubated for 30 to 60 min at room temperature, according to the methodof Mayahara ef a/. (21), in 20 ml of a medium consisting of 120 mgsodium Â¿-glycerophosphate, 2.8 ml of 0.2 M Tris-HCI buffer (pH 8.5), 9.4

mg of MgSCu, 8.0 ml of 5 mM lead citrate, 1.4 g of sucrose, 9.2 ml ofdistilled water, and 0.005% saponin (39).

After incubation, cells were postfixed in 1% osmium tetroxide, dehydrated in graded ethanol, and embedded in Epon 812 resin to form athin plate approximately 1 mm thick. After the Epon layer was separatedfrom the plastic dishes, ultrathin sections were cut parallel to the dishes.

Immunocytochemistry. Antisera prepared by Doellgast ef al. (7) were

purified on a CNBr-activated Sepharose-4B column (Pharmacia, Uppsala,

Sweden) coupled with partially purified placental ALP which was theproduct of ammonium sulfate fractionations, DEAE-cellulose ion-exchange chromatography, and Sephadex G-200 column chromatography.

Lyophilized antibodies were conjugated with horseradish peroxidase(type VI; Sigma) by the method of Nakane and Kawaoi (23). The fractionscontaining labeled antibodies were collected, and 1% bovine serumalbumin was added. For immunocytochemistry, the stock solution wasdiluted 20-fold before use. No background stains were observed at this

concentration of labeled antibodies.Immunocytochemistry was carried out by a previously published

method (39) of this La Jolla Laboratory. After fixation with periodate-lysine-p-formaldehyde solution (22), 10% normal sheep serum was added

with 0.005% saponin.Cells were then incubated in the peroxidase-labeled anti-placental ALP

IgG solution containing 1% albumin, 7% sucrose, and 0.005% saponin.The specificity of the antibody was tested by substituting normal rabbitIgG conjugated with peroxidase in place of the specific antibody. The"blocking" test was carried out by incubating cells in a solution of

unlabeled specific IgG (1 mg/ml) with 0.005% saponin before the peroxidase-labeled specific IgG incubation. As a control, unlabeled specific

IgG was replaced by 1% normal rabbit serum in the digest. To detectendogenous peroxidase in cells, the cells were maintained in phosphate-buffered saline:sucrose. After their fixation with 2% glutaralde-hyde:phosphate-buffered saline, containing 7% sucrose, the cells wereincubated in Graham-Karnovsky medium (containing, per liter: NaCI, 8g:

KCI, 0.2g; KH2PO4, 0.2g; Na2 HPO,, 1.13 g (15).

Radioimmunoassay of Placental Proteins

fi-HCG was measured with the use of reagents and proceduresprovided with the li-HCG kit (American Diagnostics, Newport Beach,Calif.). The lower limit of the fi-HCG assay was 5 mlU/ml.

HPL was measured with the use of reagents and procedures providedwith the HPL kit (Cambridge Nuclear Radiopharmaceutical Co., Billerica,Mass.). The range of sensitivity of the kit is 0.05 to 2 g/ml. The SPiassay was performed using a previously described radioimmunoassay(8). The lower limit of sensitivity of this assay was 0.3 ng/ml.

RESULTS

Cell Culture

Establishment of SKG-llla and SKG-lllb Cell Lines

Culture of the present lines was started on July 26, 1978, inplastic dishes. After a few days in the stationary period, proliferation of the tumor cells was observed, but no contaminationby fibroblasts. The monolayer cultured cells appeared to beepithelial in shape with a pavement-like arrangement. As the

cells became more dense, no contact inhibition was observed,and frequent cell overlapping was noted. On August 28, 1978,pure epithelial cells were transferred to a closed culture system,and medium was changed at least once a week and passedupon becoming confluent.

About 5 months after primary culture, the cells separatedspontaneously into 2 morphologically different strains, designated SKG-llla and SKG-lllb. SKG-llla cells (Fig. 2a) showed a

rather uniform polygonal shape with a relatively small amount ofcytoplasm. The nuclei revealed neoplastic features such as bizarre aggregation of chromatin granules, irregular thickening ofthe membrane, and rather large nucleoli. Multinucleated cellswere observed more frequently. Meanwhile, SKG-lllb cells (Fig.

2b) varied in size and shape; some cells were polygonal withabundant cytoplasm, others exhibited short-spindle shapes with

APRIL 1983 1749



S. Nozawa et al.

a rather small amount of cytoplasm. The nuclei showed a finegranular appearance with moderately hyperchromatic nucleoli.

Characteristics of the Cultured Cells

Growth Curve and Plating Efficiency (Chart 1). Two dayspostinoculation, these cells grew logarithmically; after 16 days,they reached a plateau or stationary phase. The population-doubling time determined from the growth curve of SKG-llla(passage 8) was 60 hr; that of SKG-lllb (passage 7) was 67 hr.The saturation density of SKG-llla and SKG-lllb was 4 x 10scells/sq cm and 1.5 x 10s cells/sq cm, respectively. Plating

efficiencies of SKG-llla and SKG-lllb were 15 and 32%, respec

tively.Chromosome Analysis (Chart 2). Chromosome examinations

revealed aneuploidy, and there was a wide distribution in thechromosome number with the modal number of both cell linesbeing 42.

PAS Stain. In SKG-llla cells PAS stained droplets of intracy-

toplasmic glycogen which were digested by amylase (Fig. 3).None were present in SKG-lllb cells. These results were also

similar in the heterotransplanted tumors.Heterotransplantation. Injection of SKG-llla or Illb cells into

nude mice resulted in tumor formation. Histologically, the heterotransplanted SKG-llla tumors (Fig. 4a) consisted of clear cells

which resembled the clear components of the original tumor,while SKG-lllb tumor (Fig. 46) revealed moderately differentiated

epidermoid cancer which also resembled major components ofthe original tumor.

Cell SKG-Ha (PDL No.8)

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Days of CultureChart 1. Growth curve of SKG-llla and SKG-lllb cells at eighth and seventh

transfer generations, respectively. The population-doubling (POL) time determinedfrom the growth curve of SKG-llla was 60 hr, and that of SKG-lllb was 67 hr.

Cell SKG-li (PDL No 8)

^JIh,, , , â€žiiiiilllllLLi.,.,,.,0 40 50 60 70 80 90 W0120""161Cell

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No. of Chromosome

Chart 2. Distribution of the chromosomal number of SKG-llla (eighth transfergenerations) and SKG-lllb (seventh transfer generations). Modal number of both

cells was in the hypodiploid range. POL, population doubling.

Electron Microscopy. SKG-llla cell cytoplasm was populated

by many glycogen particles (Fig. 5a). Chromatin clumps andlarge irregular nucleoli were seen in highly indented nuclei. SKG-

lllb had tonofilaments and desmosomes (Fig. 5Â£>).These resultswere also the same in the heterotransplanted tumors.

Electrophoresis for G-6-PD. The electrophoretic patterns observed are shown in Fig. 6. The G-6-PD of both SKG-llla andSKG-lllb cells migrated anodally to a position (type B) less

advanced than that of HeLa cells (type A) (Fig. 6).

ALP of Cultured Cells

Biochemistry (10, 11). Tests commonly used for characterization of term-placental isoenzyme are based on the resistanceof the enzyme to heat inactivation at 65Â°.All other isoenzymes

are rapidly inactivated at this temperature. Significant inhibitionof the enzyme by L-phenylalanine is another characteristic. Thetissue-unspecific or early-placental isoenzyme form is distinguished from the term-placental-type isoenzyme by marked heatand L-levamisole sensitivity. The small intestine-type isoenzymeis characterized by moderate heat and marked L-phenylalaninesensitivity. Term-placental isoenzyme is differentiated from Na-gao enzyme, which is also heat resistant and L-phenylalaninesensitive, by L-leucine sensitivity. Nagao isoenzyme is moresensitive to L-leucine.

Chart 3 demonstrates the results of experiments in which acomparison was made of the effects of heat treatment at 65Â°

and of various concentrations of the inhibitors, L-phenylalanine,L-levamisole, and L-leucine on enzyme extracts from SKG-llla,SKG-lllb cells, term placenta, and liver.

Heat treatment of both SKB-llla cell enzyme and term-placental enzyme for up to 15 min at 65Â°failed to cause significant

loss of activity, and the inhibitor profile of both enzymes subjected to L-phenylalanine (1 to 10 mw), L-levamisole (10~3 to 10~1

mw), and L-leucine (1 to 10 HIM)was almost identical. Meanwhile,ALP of the SKG-lllb cells was moderately heat sensitive and wasalso partially inhibited by L-phenylalanine and L-levamisole, and




Heat (65 ) Phenylalanine

Srnin ISinin

Levamisole

Iff3 5XIO-3 IO-2M

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Chart 3. Biochemical effects of heat treatment at 65Â°and of various concentrations of the inhibitors, L-phenylalanine. L-levamisole, and L-leucine on ALP extractsfrom SKG-llla (O), SKG-lllb cells (A), term placenta (â€¢),and liver (D). Heat treatmentof both SKG-llla cell enzyme and term-placental enzyme failed to cause significant

loss, and the amino acid inhibitor profile of both enzymes was almost identical.Also, ALP of the SKG-lllb cells was moderately heat sensitive and was also

moderately inhibited by amino acids.

the degree of heat and amino acid sensitivity was between thoseof term-placenta and liver isoenzyme. The sensitivity of bothSKG-llla and SKG-lllb ALP to L-leucine was greater than term-

placental isoenzyme. The average values of duplicate or triplicateexperiments of the ALP specific activity of SKG-llla cells andSKG-lllb cells were 3.7 and 1.4 nmol per mg protein per min,

respectively.Electrophoresis. The isoenzyme profile of the SKG-llla and

SKG-lllb cells studied with slab gel acrylamide electrophoresis isillustrated in Fig. 7. The doublet isozyme band of SKG-llla ALPwas retarded by anti-placental ALP antiserum but not by anti-liver antiserum. Under butyrate induction, a heat-sensitive "liver"

determinant isozyme becomes prominent. This phenomenon isto be described in another publication4.

Enzyme Cytochemistry by Light Microscopy. In order todetermine whether ALP of the cultured cells was expressed byall cells or by a subpopulation component, the enzyme cytochem-

ical staining of the cultured cells in the confluent condition wasperformed with or without heat inactivation, as well as in thepresence or absence of the differential amino acid inhibitors, L-or D-phenylalanine and L-levamisole.

Most of the SKG-llla cells expressed moderate amounts of

activity, but some of them evidenced increased amounts ofactivity (Fig. 8a). ALP activity of the majority of cells persisted

4 S. Nozawa, E. Engvall, S. Kano, S. Kurihara, and W. H. Fishman, Sodiumbutyrate modulation produces concordant expression of "early placental" alkaline

phosphatase, pregnancy-specific (i,-glycoprotein and human chorionic gonadotro-pin beta sub-unit in a newly established uterine cervical cancer cell line (SKG-llla),submitted for publication.

Uterine Cancer Cells with Regan ALP, ÃŸ-HCG,and SPi

after heat inactivation (Fig. 80). L-Phenylalanine sensitivity was

determined by comparing the difference in ALP staining afterincubation in the presence of L-phenylalanine or o-phenylalanine.If L-phenylalanine was present in the activity staining mixture,the activity of most cells of SKG-llla cells was inhibited (Fig. 8c),

but a few of the cells retained their weak activity, while the Disomer of phenylalanine caused only a slight inhibition of theactivity. Also, the inability of L-levamisole to inhibit enzyme

activity was confirmed (Fig. 8d).A small number of SKG-lllb cells showed ALP activity, and

ALP staining of a few SKG-lllb cells remained after the heatinactivation test and L-phenylalanine and L-levamisole treat

ments.These biochemical and enzyme cytochemical data suggest

that the majority of the SKG-llla cells had heat-stable, L-phenyl-alanine-sensitive term-placental ALP or Regan isoenzyme, andSKG-lllb cells, although the number of ALP-positive cells wasvery small, had both early and term-placental isoenzymes.

Enzyme Cytochemistry by Electron Microscopy. The reaction deposits were found to be granular and were visible mainlyon the plasma membrane (Fig. 9a), but the intensity of theenzyme reaction was relatively uneven. When 0.005% saponinwas used in the experiment, reaction products were also observed on such intracellular organelles as the rough endoplasmicreticulum, mitochondria, Golgi apparatus, and nuclear membrane(Fig. 9a). The intensity of reaction deposits on intracellular organelles was rather uniform compared to those of the plasmamembrane.

Immunocytochemistry. The immunocytochemical reactionwas observed on the plasma membrane of cultured cells as"wire-netting" like staining pattern (Fig. 10a). The control test

using normal rabbit IgG conjugated with peroxidase instead ofthe peroxidase-labeled anti-placental ALP IgG resulted in nega

tive staining. When the cell surface antigen was blocked withspecific IgG before the peroxidase-labeled specific IgG incuba

tion, the intensity of the reaction products was strikingly inhibited(Fig. 10o), whereas when the specific IgG was substituted withnormal rabbit serum as the control of the blocking test, thereaction was not at all inhibited, and a wire-netting pattern was

also observed on most of the cells. Endogenous peroxidase wasnegative in all cells.

Placental Proteins of the Cultured Cells

Immunoreactive 0-HCG, HPL, and SPi was absent in fresh

culture medium containing 10% fetal calf serum. However, theconcentration of /3-HCG in SKG-llla and SKG-lllb culture media

once the cells reached the confluent stationary phase was 5.0and 4.4 mlU/mg protein, respectively.

Under all the experimental conditions, the HPL concentrationcould not be detected by radioimmunoassay.

SKG-llla cells secreted SPi (0.7 ng/mg protein) in the culturemedia at confluency, but it could be identified in the SKG-lllb

culture media only in minimal amounts. The comparison of manycharacteristics of SKG-llla and SKG-lllb is summarized in Table

1.

DISCUSSION

The original tumor taken for primary culture was diagnosed asa moderately differentiated epidermoid cancer partially mixed

APRIL 1983 1751



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with "clear-cell epidermoid cancer" (27). However, the lattermust be distinguished from "glassy-cell carcinoma" (1,19) which

is considered to be a poorly differentiated mixed adenosquamouscancer. However, (a) no glandular structures were found in theregion of clear-cell components which occupied only a very smalllimited area of the original tumor; (b) in the transition areas fromepidermoid to clear-cell cancers, an intermediate cell form between these 2 was often observed; (c) clear cells often possessed abundant intracellular glycogen; and (d) an apparentsquamous differentiation in the form of dyskeratotic cells wasrecognized in the epidermoid cancer cells. Therefore, our diagnosis seems to be quite correct.

SKG-llla cultured cells in which glycogen was demonstratedby PAS stain and electron microscopy produced a heterotrans-planted tumor in nude mice which resembled very closely theclear-cell components of the original tumor; meanwhile, SKG-lllbcultured cells exhibiting many tonofilaments and desmosomes,which is one of the characteristics of squamous epithelial cells,formed the moderately differentiated epidermoid cancer in nudemice. These data suggest that SKG-llla and SKG-lllb cells arederived from the clear-cell cancer components and the moderately differentiated epidermoid cancer cell components of theoriginal tumor, respectively. Therefore, these SKG-III cell linesmight be very useful for the investigation of squamous epithelialcell differentiation.

Currently, the widespread contamination of human heteroploidcell lines by the first established human cell line, HeLa, hasbecome a crucial problem all over the world (13, 25). Nelson-Rees reported in his paper "when the cell lines exhibit (i) type A(fast) mobility for G-6-PD,(ii)phosphoglucomutase type I at locus1 and locus 3, (iii) absence of Y chromosome by fluorescentstaining, and (iv) possession of a complex of trypsin-Giemsabanded marker chromosomes present in known HeLa cells, thensuch cells regardless of designation should be considered defacto strains of HeLa." Because the electrophoresis for G-6-PD,

(which is a genetically determined enzyme and is found in A type(fast) in Negroes), of both SKG-llla and SKG-lllb cells revealed atype B (slow) mobility pattern, the possibility of a HeLa cell typecould be completely excluded.

Isoenzyme Properties. At present, ALP is subdivided into 3major isoenzymes by electrophoresis, various inhibition tests,and immunochemical methodology, e.g., tissue-unspecific (liver,kidney, bone, etc.) or early-placental isoenzyme, intestinal isoen-zyme, and term-placental isoenzyme.

Not only were there differences in the ALP specific activity(SKG-llla, 3.7 nmol per mg protein per min; SKG-lllb, 1.4 nmolper mg protein per min), but SKG-llla and SKG-lllb cells alsoshowed different isoenzyme profiles.

Although biochemical inhibition tests showed that SKG-lllaALP had almost the same characteristics as those of term-placental isoenzyme, enzyme cytochemistry clearly demonstrated a few L-phenylalanine-insensitivecells, and immunocy-tochemical staining with anti-term-placental ALP antibody demonstrated some negatively staining cells, in spite of the fact thatby enzyme cytochemical staining most of the cells at confluencyshowed ALP activity. These data suggest that some of the SKG-llla cells, although the number may be small, might have tissue-unspecific or early-placental ALP isoenzyme.

Taking all of these data into consideration, together with theimmunocytochemical staining which proved the existence of the





same protein as the term-placental ALP, we can conclude thatmost of the SKG-III ALP is the term-placental isoenzyme, but asmall amount of the early-placental ALP is also included in theSKG-III cells. Further, ALP of both cell lines showed a littlegreater L-leucine sensitivity than the term-placental ALP, which

was clearly less than Nagao isoenzyme. Therefore, we could notrule out the possibility that ALP of these cultured cells sharedproperties of Nagao isoenzyme.

Membrane Sites of ALP in SKG Cells. Up to the present,ALP has been considered to exist mainly in the plasma membrane, but its activity in the mitochondrial membrane or endo-

plasmic reticulum was also reported (4, 36). In this experiment,by using the diluted saponin step of Tokumitsu ef a/. (39), wecould demonstrate the ALP activity in such intracellular organ-

elles as the rough endoplasmic reticulum, Golgi apparatus,plasma membrane, nuclear membrane, and mitochondria. Although the exact role of saponin to permit the staining "cryptic"

ALP has not yet been clarified, it not only may help the substrateto penetrate the cell matrix but may also change the physicalconstitution of the membrane. The existence of ALP in somekinds of intracytoplasmic organelles seems to be very reasonable, because ALP is one of the glycoproteins, and most membrane glycoprotein components are believed to be synthesizedon the endoplasmic reticulum, modified in the Golgi apparatus,and transported to and incorporated into the surface plasmamembrane.

Concordant and Discordant Gene Expression. Since Fish-man's first report (11), the term-placental ALP or Regan isoen-

zyme has been considered as one of the oncotrophoblasticproteins. However, it is also true that many tumors which includeuterine cervical dysplasia or carcinoma in situ or invasive cancer(30, 31) have heat-labile tissue-unspecific (liver or bone) isoen-zyme. This tissue-unspecific isoenzyme was previously found tobe expressed by early-pregnancy placenta of less than 10 weeksgestation (10, 35) and in HeLa TCRC-2 cells. In this paper, weprefer to use the term "early-placental ALP." Therefore, as some

of the tumor ALP might correspond to early-placental ALP, early-

placental ALP could be considered as one of the oncotrophoblastic proteins. If so, SKG-III cells express at least 4 kinds ofoncotrophoblastic proteins such as early-placental ALP, term-placental ALP, 0-HCG, and SP,.

Braunstein ef al. (5) reported that the secretion of HCG andSP, may be regulated by a similar control mechanism during thefirst trimester of pregnancy. When we consider that early-placental ALP is also an early-placental gene product, it may be possible

to demonstrate the coexpression of these 3 kinds of earlytrophoblastic gene products of cancer cells using such genemodulators as sodium butyrate or prednisolone, both of whichhave been shown to be modulators of oncotrophoblastic geneexpression (6, 37). This matter is the subject of a separatecommunication."

It is suggested that, in contrast to HeLa, the SKG-III cell line

may serve as a more representative model system available forstudies of the mechanisms of regulation of these oncodevelop-

mental genes in tumors of gynecological origin.

ACKNOWLEDGEMENTS

The authors greatly appreciate Dr. E. Engvall for help with the SP, determinationsand Dr. S. Tokumitsu, Dr. W. C. Hanford, and Dr. R. Jemmerson tor many helpfuldiscussions and suggestions at La Jolla Cancer Research Foundation.

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Fig. 1. Most of the original tumor was populated by moderately differentiated dark epidermoid cancer cells, but minor components of clear cells with PAS-positivecytoplasm (arrow) were also present. PAS, x 200.

Fig. 2. In a, SKG-llla showed a rather uniform polygonal shape with relatively little cytoplasm. The nuclei revealed more neoplastic features than did those of SKG-lllbcells. In 6, SKG-lllb cells varied in size and shape. Some cells were polygonal with abundant cytoplasm, and others were short-spindle shaped, with rather small amounts

of cytoplasm.Fig. 3. Intracytoplasmic glycogen droplets seen only in SKG-llla cells. PAS, x 200.

Fig. 4. a, heterotransplanted SKG-llla tumors consisting of clear cells which resembled the minor components of the original tumor; b, heterotransplanted SKG-lllbtumors revealing moderately differentiated epidermoid cancer which also resembled major components of the original tumor. H & E, x 200.

Fig. 5. Transmission electron microscopy, a, SKG-llla cells, characterized by many glycogen particles (GL) in the cytoplasm. Chromatin clumps (CC) were seen innuclei, x 19.200 Â£>,SKG-lllb cells with a high tonofilament (TF) content in their cytoplasm and many desmosomes (DM) at the adjacent cell membrane, x 16,800.

Fig. 6. Electrophoresis of G-6-PD of HeLa cells (Lane 1) and SKG-llla cells (Lane 2). The G-6-PD of both SKG-llla and SKG-lllb cells migrated more slowly (B form)than that of HeLa cells (A form).

Fig. 7. Immunoelectrophoretic analysis of SKG-llla ALP. SKG-llla isoenzyme showed one major band (Lane 7), and it was prevented from migration by the term-placental ALP antibody (Lane 3) and not by the antiserum directed toward liver (Lane 2).

Fig. 8. Enzyme cytochemical characterization of SKG-III ALP. a, black precipitate in most of the SKG-llla cells, indicative of activity. In b, after heat inactivation (65Â°for 30 min), heat-stable ALP activity persisted. In c, in the presence of u-phenylalanine, an obvious inhibition is seen. ALP in most SKG-llla cells resembled the Reganisoenzyme, but a few of the cells retained their weak activity. In d, after a L-levamisole inhibition test, ALP in most cells remained, x 200.

Fig. 9. Electron microscopic enzyme cytochemistry for SKG-llla ALP. In a, reaction products were mainly observed on plasma membrane (long arrows), but weakactivity was observed on the nuclear membrane (snort arrows), x 7,800. In b, ALP activity was also observed in Golgi apparatus (GO), endoplasmic reticulum (ER), andmitochondria (MT). x 11,000.

Fig. 10. Immunocytochemical staining of ALP in SKG-llla cells with anti-term-placental ALP IgG labeled with peroxidase (direct method), a, immunocytochemicalreaction, observed on the plasma membrane of SKG-llla cells as "wire-netting"-like staining pattern, b, when the cell surface antigen was blocked with the specific IgG

before the peroxidase-labeled specific IgG incubation, the intensity of the reaction products was strikingly inhibited, x 50.




1983;43:1748-1760. Cancer Res Shiro Nozawa, Yasuhiro Udagawa, Hiroaki Ohta, et al.

-Glycoprotein Phenotypes1β-Subunit, and Pregnancy-specific βwith Regan Isoenzyme, Human Chorionic Gonadotropin

Newly Established Uterine Cervical Cancer Cell Line (SKG-III)

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