human chronic m yelogenous leukemia cell-line with - blood

Blood, Vol. 45, No. 3 (March), 1975 321

Human Chronic M yelogenous Leukemia Cell-Line

With Positive Philadelphia Chromosome

By Carmen B. Lozzio and Bismarck B. Lozzio

A cell-line derived from a patient withchronic myelogenous leukemia (CML) isdescribed. The new cell-line, which hasover 175 serial passages in a 3k-yr period,has the following characteristics: (1) CMLcells started to proliferate actively sincethey were first incubated in culture media.A threefold increase in the total number ofcells was observed during the first sevenpassages; the cell population increased bya factor of 10 to 20 every 7 days from pas-sage 8 through 85; from 20 to 40 timesfrom passage 86 through 150, and morethan 40 times after 150 passages. (2) Themajority of the nononucleated cells areundifferentiated blasts. (3) The karyotype

of all the cells examined show the Phila-delphia (Ph’) chromosome and a longacrocentric marker plus aneuploidy. The

Giemsa-banding studies identified the Ph’chromosome as a terminal deletion of thelong arm of chromosome 22:del(22)(q12)and the long acrocentric marker as anunbalanced reciprocal translocation of onechromosome 17 and the long arm of onechromosome 15. (4) The CML cells do notproduce immunoglobulins, are free ofmycoplasma, Epstein-Barr virus, andherpes-like virus particles. (5) CML cellshave no alkaline phosphatase and myelo-peroxidase activities and did not engulfinert particles. (6) Cultured CML cellsprovide a constant source of a specific an-tigen. This CML cell-line represents aunique source of CML cells with meaning-ful indicators of malignancy for clinicaland experimental studies.

A TTEMPTS TO ESTABLISH permanent cell-lines derived from the blood

of patients with chronic myelogenous leukemia (CML), having the Phil-

adelphia (Ph’) chromosome marker, have been consistently unsuccessful,’� and

cultures showing the Ph’ chromosome for several months5 have lost the Ph’

chromosome after serial subcultivation for longer periods of time.4 In all these

cultures,’5 the leukemic cells were replaced by immunoglobulin-producing

(lymphoid) cells. Recently, the growth and maturation of CML cells was stud-

ied in liquid media. Bone marrow and peripheral blood cells from seven pa-

tients with Ph’-positive CML were cultured in diffusion chambers for up to 45

days. The Ph’ chromosome was found in only two cultures.6 The Ph’ chromo-

some was also found in short-term soft-gel media cultures.7

Preliminary reports from our laboratory showed persistence of the Ph’ chro-

mosome 20 mo after establishment of a CML cell-line from a pleural effusion

of a terminal case in blastic crisis.� These cells have continued proliferating

very rapidly and maintain their positive Ph’ chromosome and a long acrocentric

rearrangement after continuous culture for 3� yr and over 175 serial passages.

The purpose of this report is to describe the origin, culture method, and

characteristics of this unique cell-line.

From the Laboratories of Cytogenetics (CBL) and Spleen Pathophysiology (BBL). The University

of Tennessee Memorial Research Center and Hospital, Knoxville, Tenn. 37920.

Submitted June 14. 1974; accepted September 22. 1974.

Supported in part by American Cancer Society Grant CI 87B and Grant FR 5540.

Address for reprint requests: Carmen B. Lozzio, M.D., The University of Tennessee Memorial

Research Center and Hospital. Knoxville. Tenn. 37920.

© 1975 by Grune & Stratton, Inc.

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322LOZZIO AND LOZZIO

MATERIAL AND METHODS

Case History

The cell line described herein was derived from leukemic cells obtained in December 1970 froma pleural effusion of a 53-yr-old female suffering from CML for about 4 yr. She had been treatedwith busulfan for 3 yr and pipobroman for I yr. In October of 1970, all peripheral blood cells

showed a positive Ph’ chromosome and a clonal evolution of the leukemia with a new cell-line

of45 chromosomes and a translocation D/E. In November of 1970, a splenectomy was made.Soon after surgery the patient underwent blastic crisis, and the malignant cells accumulated astumor masses of highly undifferentiated cells through most tissues of the body and produced

pleural effusion. Nine days prior to the patient’s death a sample of the pleural fluid was ob-

tamed and used for chromosome analysis in short-term cultures, and to start the long-term cul-

tures. A positive Ph’ chromosome and a long acrocentric chromosomal rearrangement were ob-served in all cells. The aggressiveness and extensiveness of the malignant infiltrate suggestedmarked alteration of the original leukemic cells, and the presence of a positive Ph’ chromosome

in addition to the lack of structural patterns of the tumor masses provided evidence of the myeloid

origin of these neoplastic cells.

Establishment of a CML Cell-line in Suspension Cultures

The pleural fluid was collected with heparin and aliquots containing 15-20 x 106 cells eachwere diluted with Eagle’s minimal essential medium (MEM) plus additional amino acids and 15%

fetal bovine serum. All cultures were incubated at 37’ in a humidified atmosphere with constantflow of 5% CO2 in air. The cells grew as a free-floating cell suspension since they were first in-cubated in liquid cultures. Serial subcultivations of aliquot samples were carried out weekly

after determination of the total number of cells per culture. The cell-line was named CM L- I,

K-562.

Freezing and Thawing of Cultured Cells

Samples of cells were frozen at various passages using Eagle’s medium supplemented with 5%

glycerol. The cells were frozen at l’C per mm and the ampules stored in liquid nitrogen. The

ampules were thawed rapidly, and the cells were suspended in culture medium after determination

of their viability with 0.5% trypan blue.

Colony Growth in Agar-gel and Isolation of Cell Clones

The method of Robinson and Pike10 was followed with slight modifications. The plates, made up

of only one layer of CMU cells mixed with 3 ml of semisolid agar diluted with McCoy’s 5A medium

were incubated at 3TC under controlled pH and humidity for 12-14 days. The number of colonies

per plate and the plating efficiency of the CML cell-line were determined at various passages.

The plating efficiency was also investigated in plates containing a feeder layer of peripheral leuko-cytes (2 x 106) from normal humans.

The agar-gel method was used also to isolate clones of cells derived from single cells. The

clones were isolated by picking individual colonies which were transferred to Leighton tubeswith I ml of medium. Each colony grew as a suspension culture and was transferred to culture

bottles with increased amounts of media until the cell density was similar to the original cell

line K-562. Each clone was maintained by weekly passage. After chromosome analysis of all the

clones, samples from each clone were stored frozen in liquid nitrogen.

Cytogenetic studies

Chromosome preparations were made at various passages according to the procedure of Moor-

head et � These smears were stained with Giemsa dye after hydrolysis with I N HCI. Chro-mosome analyses were also performed in smears stained by the Sumner and Evans’ Giemsa-

banding t��hfliqu�,�2 and by the fluorescence method of Caspersson et al.13The length of the cell cycle was determined at various passages by the technique of labeled

mitosis of Quastler and Sherman.14 A suspension containing 10 cells/mI was pulse labeled for




MYELOGENOUS LEUKEMIA CELL-LINE 323

15 mm with 1 zCi/ml of 3H-thymidine (specific activity, 1.9 Ci/mM: Schwarz/Mann, Orange-

burg, N.Y.).

These cells were centrifuged, rinsed with balanced salt solution, and resuspended in freshculture medium without the isotope. Samples of 10 ml were incubated at 37’C until fixation at

30 mm, 1,2,3,4,5,7,9,1 1,13,15,17,19,21,23, and 25 hr after pulse labeling with 3H-thymidine.

The radioautographs were exposed for 10 days, developed with D I I, stained with Giemsa dye.The per cent of labeled versus unlabeled metaphases was determined.

Cytology

The morphology of CML cells growing either in liquid or semisolid media was studied peri-

odically on smears stained with Giemsa dye.

Enzyme Studies

The Graham-Knoll peroxidase reaction for general differentiation and cytochemical demonstra-tion of alkaline phosphatase according to Gomori technique’5 were performed on smears of

normal peripheral leukocytes (control) and cultured CML cells.

Viral Testing

Cultured cells were tested for Epstein-Barr virus (EBV) genome and EBV-associated nuclearantigen referred to as ENBA.’6 These tests were kindly made by Dr. Gertrude and Dr. Werner

Henle of the Division of Virology, Childrens Hospital, University of Pennsylvania, Philadelphia,

Pa. Mycoplasm (PPLO) contamination was tested using media and supplements from Microbio-logical Associates, Bethesda, Md., following the method of Hayflick and Stanbridge,’7 and Hay-

flick.18 The media and cells were monitored for PPLO continuously. Tissue culture reagents were

also tested for mycoplasm before use following the procedures of Barile and Kern.19

Immunoglobulin (Ig) Determination

The technique of Mancini et al.20 was followed throughout with slight modifications. Eachplate contained agar impregnated with goat monospecific antiserum. Cells, cultured for 7 days,

were centrifuged. The concentration of Ig was determined in nonconcentrated and 10 x con-

centrated media. The media were concentrated by ultrafiltration. A cell pellet containing

20 x 106 CML cells was washed once with cold saline disrupted by sonic vibrations, and an

aliquot assayed for 1g.

Phagocytosis

The ability of CML cells to ingest polystyrene particles was determined following the technique

of Cline and Lehrer.21 The culture medium was the same used to grow the cells in suspension.Polystyrene particles (1.3-1.7 .t in diameter) were added to tissue culture medium at the rate

of 1 mg/mI. Five milliliters of the suspension were mixed with either 2 x 106 CML or normalIeukocytes (control) contained in 5 ml of medium. The cells and polystyrene particles were incu-

bated for 1 hr at 37’C with shaking. Intracellular particles were studied by phase-contrastmicroscopy.

RESULTS

Growth Characteristics in Free-floating Suspension Cultures

The CML cells started to proliferate actively as soon as they were incubated

in culture medium. No latent period was observed, but the rate of growth varied

at various passages as follows: one to three times increase per passage during

the firstseven passages, 10-20 times from the 8th to the 85th, 20-40 times from

the 86th to the 150th, and 40-70 times from the 150th to the 175th passage. A

corresponding decrease in the mean generation time was observed. This was

approximately 50 hr during the firstseven passages, and 10-12 hr after passage




324 LOZZIO AND LOZZIO

85. The decrease in the total cell cycle was probably due to a shortened , stage

as seen in cells studied at passage 96. At this passage, the total cycle lasted 12

hr, including a period of DNA synthesis (5) of 8 hr, a postsynthetic stage G2

of2 hr, and a 0, plus mitotic period of2 hr.

Cytologic Features ofCML Cells

The morphologic characteristics of CML cells at different passages of the

culture were those of highly undifferentiated cells of variable size (Fig. IA).

The majority ofthe round or oval cells measured 14-17 z in diameter, approx-

imately 25% ofthese cells had 20-35 �, 10% had 7-8 � and 5% 40-55 s. Poly-

ploid cells were frequently seen. There was not a sharp delimitation between the

nucleus and cytoplasm in the majority of the cells. The cytoplasm was in-

tensely basophilic, devoid of granules, and contained few small vacuoles.

Atypical promyelocytes with eccentric nucleus and scanty azurophilic granules

in the cytoplasm account for less than 2% of the cell population. Several cells,

with a diameter of 20 � or over, had deeply basophilic cytoplasmic buds which

in some cells were extremely large: one-third of the cell diameter. The nuclei

were round, slightly indented, and, occasionally, of semi-moon-like shape dur-

ing interphase. One to four nucleoli were easily discernible in each nucleolus,

regardless of the cell size. Large-sized nucleoli were often uncovered so that

their blue-violet color was very prominent. The chromatin was irregularly

granular or formed fine laced threads which stained reddish violet with Giemsa.

Numerous mitotic figures and bin ucleated cells were also seen. Another distinct

cell type was characterized by a pale blue cytoplasm and a large nucleus, con-

taining two to six blue nucleoli within long skeinlike threads of chromatin.

These cells developed and proliferated particularly well on agar (Fig. I B).

Growth and Colony-forming A bility in Agar

The number of colonies was directly proportional to the number of cells

plated, as demonstrated in triplicate plates simultaneously made with 250, 500,

and 1000 CML cells. Small round colonies containing less than 100 cells were

seen by day 5 of incubation, and colonies containing more than 2000 cells were

seen on day 14 of culture. The plating efficiency was between 50% and 60%,

with an average of 582 ± 67 (1 SD) colonies in the plates inoculated with 1000

cells without a feeder layer. In general, colonies arose from single cells, but

occasionally groups of two to three cells were present and originated even

larger colonies than those described above.

In five experiments, a feeder layer of normal peripheral leukocytes was added

to the hard agar underlayer, and CML cells were added on top with the soft

agar layer. The plating efficiency was reduced by a factor of 5-6, and no effect

on CML cell differentiation and maturation was observed.

The morphology of all cells in 156 colonies examined was nearly identical

(Fig. lB-D). Two main highly undifferentiated cell types proliferated but did

not differentiate into more mature forms of the granulocytic series. All cells

growing on agar were very large (40-60 � in diameter) and exhibited numerous

vacuoles in the narrow band of deeply basophilic cytoplasm surrounding the

nucleus.





Fig. 1. (A) Compositive photograph showing chronic myelogenous Ieukemic (CML) cells grow-ing in 7-day-old suspension cultures. (8-0) CML cells developing in semisolid agar in a 15-dayperiod. A small part of a large colony is seen in (D). Both liquid and agar cultures were madeon passage 168 of the CML cell line K-562. Giemsa stain. x 540.




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Cytogenetic Studies of the Patient Cells in Short-term Cultures

A description of the cytogenetic studies of the patient’s cells is given herein as

a control for the cultured CML cells to show their origin in clones of cells with

abnormal karyotypes. A positive Ph’ chromosome was seen in all the metha-

phases studied from peripheral blood in October 1970. Two modal numbers of

chromosomes were observed: 28 cells had 45 chromosomes and 22 cells had 46

chromosomes. The cells with 46 chromosomes had a Ph’ chromosome in an

otherwise normal female karyotype, and the cells with 45 chromosomes had a

positive Ph’ and had lost one member of the group D (13-15) and one of group

E (17-18) with apparent gain of one submetacentric of group C (6-l2-X)

(Fig. 2), which was interpreted as a reciprocal translocation D/E.

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Fig. 2. Karyotype 45,XX,-D, -E,+t(D;E) observed in 24-hr cultures of peripheral blood o�-tamed from the patient 2 mo before the long-term cultures were started. Note the absence of onechromosome from group D and one from group E (probably a number 17) and the presence ofan extra C-like chromosome. The arrow indicates this chromosome, which was interpreted as atranslocation between one chromosome of group D and one of group E. The Philadelphia chromo-some (Ph’) is also identified by an arrow. Fifty per cent of the cells cultured from peripheralblood had this karyotype and the other 50% had a karyotype 46,XX,Ph1 +.




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Fig. 3. Karyotype of an hypodiploid cell showing a long acrocentric marker and a positivePh’ chromosome. This marker and the Ph’ chromosome were observed in all the cells analyzedin short-term cultures of 24 hr from the same pleural effusion used to start the long-termcultures.

The cells from the pleural effusion, obtained in December 1970, showed

marked aneuploidy with a large proportion of hypodiploid cells. Ninety-two of

one hundred cells in metaphase had a chromosome number varying from 40 to

45 chromosomes. All the cells had a Ph’ chromosome and a long acrocentric

chromosome significantly larger than the other members of group D (13-15)

(Fig. 3) and a consistent loss of one chromosome of group E (16-18).

Cytogenetic Studies in Long-term Cultures of the CML Cell-line at

Various Passages

Chromosome analyses of the cultured CML cells were performed at passages

5, 10, 20, 40, 90, 110, 144, and 171. One hundred cells were studied, and five to





ten karyotypes were analyzed from each passage. These studies revealed that all

the cells contained at least one Ph’ chromosome, plus the long acrocentric

marker described in the short-term cultures of the pleural effusion. A small

percentage of cells with three or four Ph’ chromosomes was seen, but they were

not directly related to either the age of culture or the age of the cell-line K-562.

Changes in the modal number of chromosomes, as well as appearance of new

clones with additional chromosome markers, were observed at different pas-

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Li ..� UFig. 4. Karyotype of a pseudodiploid cell with 46 chromosomes stained with the G-banding

technique. This cell was observed in long-term cultures of the cell-line CML K-562 at passage 110.The banding pattern shows that the long acrocentric chromosome is probably a translocationbetween one chromosome 17 and one chromosome number 15, and the Philadelphia chromosomeis a deletion of half of the long arm of one chromosome number 22. Note the presence of anextra chromosome number 1 and one number 22 and the absence of one chromosome number11 andonenumberl2.




MYEIOGENOUS LEUKEMIA CELL-LINE 329

sages. By the passage 5, the cells maintained their hypodiploid mode (40-45),

and the majority of the cells have lost one group C chromosome (6- 12-X), while

the minority of the cells have lost a chromosome number 1, or one of the

chromosomes ofgroup D (13-15), E (16-18), or F (19-20). On passages 10 and

20, the majority of the cells had an hyperdiploid modal number of 50-52

chromosomes. At passage 84, two sublines were started by performing serial

passages at different days of the week. At passage 1 10, karyotypes of the two

sublines were studied and revealed a predominant near triploid mode (69-73)

in one subline and a near tetraploid mode (90-96) in the other subline. A new

marker chromosome appeared in the near tetraploid subline. These cells had a

chromosome number 2 with a significant elongation of the long arm (2q+)

in addition to the long acrocentric and one to four Ph’ chromosomes.

To demonstrate if cells growing on agar had the same chromosome markers

as those cultured in liquid medium, several individual colonies were removed

from the agar and cultured in suspension. The cells isolated from semisolid

agar showed the same Ph’ chromosome and the marker present in the blasts

proliferating in the pleural effusion before the suspension cultures were started.

Ten clones were isolated: seven were aneuploid with a predominant near

triploid mode (67-73), two had two modal numbers, and one clone had hyper-

diploid cells with a modal number of 52 chromosomes. Repeat chromosome

analyses of the hyperdiploid clone showed that near triploid cells had over-

grown the hyperdiploid cells 4 mo after the clone was established. All the karyo-

types analyzed from these clones had one to four Ph’ chromosomes and a long

acrocentric marker.

Chromosome analyses with the G-banding technique (Fig. 4) were performed

at passages 110 (January 1973), 144 (September 1973), and 171 (April 1974) of

the original line. The hyperdiploid clone was studied at passages 8 and 21 after

cloning. Q bands were investigated at passage 144. The G- and Q-banding

studies demonstrated that the Ph’ chromosome is a terminal deletion (del) of

the segment distal to band 12 of the long arm (q) of one chromosome number

22. Therefore, the Ph’ chromosome may be designated as a del(22) (q12), ac-

cording to the nomenclature established at the Paris Conference.22* The band-

ing studies also showed that the long acrocentric marker is an unbalanced

reciprocal translocation between one chromosome 17 and one chromosome

number 15 (Fig. 5). This translocation involved the breakage of the long arm

(q) of chromosome 17 at the band number 24 and the long arm of chromosome

15 at band number 21. Therefore, the translocation is reported as t(15;17)

(q21;q24) according to the Paris Conference.22 This translocation originated

in vivo as the result of clonal evolution and persisted in vitro for over 175 serial

passages. Other chromosomes, including chromosome number 9, had normal

G-bands in contrast to reports from other cases with CML,23-24 but new rear-

rangements appeared as the result of in vitro clonal evolution.

In summary, the banding studies have identified several chromosomal struc-

*Explanation of terminology established at the Paris Conference: del, deletion; q, long arm;

t, translocation; for additional explanation of the chromosome aberrations reported see Fig. 5.





t(15;17)(q21;q24) �

17

15 �hIIftb*bI21 J� #{149}I6’�O*

22

Ph1:deI(22)(q12)fi� #{149}0 A frFig. 5. Partial karyotype of six mitotic cells from passage 171 of the CML cell-line stained

with the Giemsa-banding technique and diagrammatic representation of the Giemsa bands. The

first row shows the translocation of the distal half of the long arm of one chromosome 15 tothe end of the long arm of chromosome 17. This translocation was seen as a marker in all thecells studied in the short-term cultures and in all the cells analyzed at various passages of thiscell line. The normal chromosomes numbers 17, 15, 21, and 22 are shown on the second, third,fourth, and fIfth rows. The law row demonstrates the terminal deletion seen in all the Ph�chromosomes.

tural aberrations, two of them present before the cultures were started, and

others which were developed during in vitro subcultures.

The modal number of chromosomes has changed in vitro with spontaneous

evolution of new sublines and clones with faster growth rates and increased

numbers of chromosomes. Thus, clones containing two cell types are over-

grown by the cell type with the largest chromosome number. For this reason,

repeated cloning of the original cell-line in agar plates and selective isolation of

clones with pseudodiploid karyotypes are required to maintain a source of

pseudodiploid CML cells with characteristic chromosome aberrations available

for experimental use.





Enzyme Studies

Normal granulocytes exhibited a strongly positive peroxidase and phospha-

tase reaction, whereas cultured CML cells had consistently negative reactions

in ten slides containing several hundred cells each.

Viral Antigens

The CML cell-line was found to be free of the EBV-associated nuclear anti-

gen which is present in most lymphoblastoid cell-lines carrying the EB viral

genome. Routine tests for mycoplasma were always negative.

Immunoglobulins

A search for Ig in the CML cells and culture media failed to reveal any

detectable amount of IgA, IgG, and 1gM (Fig. 6).

Phagocytic Activity

Monocytes were more efficient than neutrophils to engulf polystyrene par-

ticles. After 60 mm of incubation, a greater number (six or more) of polystyrene

Fig. 6. Quantitative determination of immunoglobulins (lg) by single radial diffusion.(A-C) Reference sera: lgG, 4, 10, and 20 iig/ml; lgM, 0.32, 0.80, and 1.60 pg/mI; and IgA,

0.8, 2.0, and 4.0 pg/mI. (0-F) Test samples: (D) Undiluted culture medium, (E) tenfold con-centrated culture medium, and (F) disrupted CML cell suspension containing 20 x 106 cells/mI.Media and cells were from 5-day-old cultures of the CML cell-line K-562. No detectable amounts ofIgG, 1gM, and IgA could be demonstrated in the media and the CML cell pellet at several passagesof the culture.





were seen in monocytes, whereas two to five particles were observed in neutro-

phils and eosinophils. In marked contrast, CML cells cultured in liquid or semi-

solid media did not ingest polystyrene particles. Also, none of the large CML

mononuclear cells developing in semisolid medium phagocytize agar.

DISCUSSION

We described herein a unique cell-line derived from a pleural effusion of a

patient with CML. As far as we are aware, the CML-l, K-562 is the first

permanent cell-line available with a persistent positive Ph’ chromosome after

prolonged in vitro cultivation. We believe that the successful establishment of

this cell-line is due to the active proliferative capacity of highly undifferentiated

cells obtained from the patient at the terminal stage of an acute blastic crisis.

These cells were derived from a clone with a Ph’ chromosome and a new

chromosome rearrangement involving the long arm of one chromosome 17.

Rearrangements ofthe long arm ofchromosome 17 have been reported in CML

patients25’26 in blastic crisis and might be another cause of the increased growth

rate.

The leukemic origin of the cultures derived by others from CML leukemic

blood4 may be the result of differential in vitro proliferative capacity of normal

and leukemic cells. Indeed, the critical factor could be the absence of normal

cells in the pleural effusion used to start our cultures. The malignant CML

blasts (K-562) proliferated well in suspension cultures and retained the original

karyotype because they were derived from a clone of malignant cells with high

proliferative capacity which were already growing as a cell suspension in the

pleural fluid and were not overgrown by the proliferation of normal cells, as has

occurred with most permanent cell-lines established from human leukemic

blood.4

The myeloblastic cell-line (K-562) described herein differs from the lympho-

blastoid cell lines described by others (see reference 4) in many characteristics.

The CML cells have a positive Ph’ chromosome, they lack immunoglobulins,

they are negative for EBV or herpes-like virus, and began to proliferate actively

as soon as the suspension cultures were started. In contrast, lymphoblastoid

cell-lines established at other laboratories began to proliferate rapidly after a

latent period of 4-15 wk,4’27�#{176}during which the number of cells decreased sig-

nificantly. After establishment of the lymphoblastoid cell-line, herpeslike virus

particles were detected by electromicroscopy,4’27-30’33 and most cell-lines carried

the EBV genome.4”6’3”� It is believed that an interaction between EBV and

human diploid cells is essential for the establishment of a permanent lympho-

blastoid cell-line. Also, the majority of these cell-lines synthesize immuno-

globulins.4’30’3536 Preliminary electron microscopic examinations of CML cells

(K-562) fail to show herpes-like virus particles. Therefore, the absence of EBV

and herpeslike virus and the failure of this CML cell-line to produce immuno-

globulins are three important differential characteristics that indicate that the

line (K-562) is not another culture of lymphoblastoid cells. Furthermore, the

presence of the Ph’ chromosome conclusively demonstrates that this cell-line

is of CML origin.

As expected, the undifferentiated CML cells were devoid of alkaline phospha-





tase and peroxidase activity, and lacked the capacity of ingesting inert particles,

a property of more differentiated granulocytic cells and macrophages.

The CML cell-line has been found to be a continuous source of a specific

antigen.37 Work in progress indicates that a rabbit anti-CML K-562 serum ad-

sorbed on normal granulocytes is cytotoxic for cultivated CML cells and for

CML cells obtained from untreated patients with this disease. The cross-reactiv-

ity of the rabbit anti-CML serum with cultured CML cells and those obtained

from patients with CML before treatment suggests a common antigen, which

may be of value for the detection, prognosis, and/or immunotherapy of CML.

In summary, this new CML cell-line derived from undifferentiated mye-

logenous leukemic cells with karyotypic clonal evolution is the first human

CML cell-line available which has retained meaningful indicators of malig-

nancy: chromosome aberrations, cloning efficiency on agar, and a specific anti-

gen(s) after prolonged cultivation. Therefore, it represents a unique source of

human CML cells for experimental and clinical studies.

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2. Belpomme D, Le Borgne Dc Kaouel Ch,

Paintrand M, Grandjon D, Venuat AM, de

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#{224}prespassage en ligen#{233}econtinue. Rev FrEtud Clin Biol 14:848, 1969

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1975 45: 321-334

CB Lozzio and BB Lozzio chromosomeHuman chronic myelogenous leukemia cell-line with positive Philadelphia

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