Mouse myelomas and lymphomas in culture

Experimental Cell Research 60 (1970) 61-77

MOUSE MYELOMAS

AND LYMPH0

K. HORIBATA The Salk Institute for Biological

AS IN CXJLTU

and A. W. HARRIS

Studies, Post Office Box 1809, San Diego, Calif. 92112, USA

SUMMARY The establishment and growth characteristics of nine different lonn-term continuous in vitro cell lines originating from t%e myeloma anad two lymphoma tumor lines of independent origin in BALBic or C3H mice. is described. Different culture lines were initiated bv somewhat different methods but they now all grow in fortified Eagle’s medium supplemented-with horse serum as stationary suspension cultures with doubling times between 16 and 26 h. Some of the lines are near tetraploid and contain marker chromosomes while others are diploid or near-diploid. Several of the myeloma lines synthesize and secrete immunoglobulin while others, probably as a consequence of mutation, do not. The two lymphoma lines may synthesize Ig but do not appear to secrete it. Assays of the growth inhibiting activities of thymidine and hydrocortisone show that the lymphoma cells are much more sensitive than the myeloma cells. There is evidence suggesting that in these respects, as well as others, the cultured cell lines possess some of the differentiated characteristics of normal lymphocytes and plasma cells. Possible uses of the cell lines to analyze at the biochemical level the modes of genetic control of such characteristics are discussed.

The homogeneous immunoglobulins produced by plasma cell tumors (myelomas) in mice and in humans have provided the experimental material responsible for the recent breakthrough in our knowledge of immunoglobulin structure [11, 171. Amino acid sequences derived from these proteins have been employed in many recently proposed models to explain the origin of antibody molecule diversity as it occurs in immune responses [5]. The myeloma cells themselves have been used in experiments to define the mechanisms of synthesis of Ig subunits and the mode of their subsequent assembly and secretion from the cell [20, 39, 441. They shouid also be useful in studies of the genetic control of Ig synthesis. Some work along these lines with mutant myelomas has already 1 Present address: The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital Post Office, Victoria 3050, Australia.

been reported 14,40, 41, 431.The ~dva~~a~;es of myelomas over normal plasma these investigations are of course that sent only the former can be obtained as a growing cloned cell population, a fact which relates to the regulation of division as a stem cell goesdown the pathway to a specialized dead-end state [4, 41. degreesof experimental rnani~~lab~~~tywould be acquired if myeloma cells were cultivated continuously in vitro instead of as tumors in vivo. Another cell type vitally involved in immune responsesis the lymphocyte 1121which can also be obtained as neoplastis variants or lymphomas. It is probable that ~yrn~~ornas can be used as models for the study of the physiology, biochemistry and genetic control of specific lymphocyte functions just as the myelomas can be for plasma cell functions. With these objectives in min

62 K. Hovibata h A. W. Harris

sidered it important to establish tissue culture lines derived from myelomas and lymphomas. A few attempts to grow myelomas of human and mouse origin have been made by others [27, 28, 291 and some have been successful [23, 25, 301.We have established a number of continuous cultured cell lines derived from myelomas and lymphomas, most of them originally induced in BALB/c mice by treatment with a mineral oil [4, 311. Some of the cell lines display properties characteristic of their presumed normal cells of origin. This paper describes the establishment in vitro of nine such cell lines and derivatives of them, their growth characteristics and some of their specialized activities.

Isolation of clones in the spleen Numbers of viable tumor cells ranging between lo3 and lo6 were injected intravenously into young adult host mice and the animals were examined for the formation of spleen nodules lo-14 days later. Well isolated colonies were dissected out and each transplanted subcutaneously into a mouse.

Cell culture environment All cultures were grown in disposable plastic Petri dishes (Falcon Plastics, Los Angeles, Calif., although several other manufacturers’ products were equally suitable), usually of the bacteriological type.. The medium consisted of fortified Eagle’s medium (FEM) [46] containing 0.37 % NaHCO,, supplemented with heat-inactivated (56°C 30 min) horse, bovine or fetal bovine serum (Hyland Laboratories, Los Angeles, or Grand Island Biological Company, Oakland, Calif.) and other materials as designated below. Cultures were incubated at 37°C in a humidified atmosnhere of about 13 % CO, in air.

Cell counting and viability

MATERIALS AND METHODS Myelomas MOPC21 and Adj PC-5 are transplantable plasmacytomas of BALB/c origin, producing immunoglobulins of type yG, and YG,,, respectively [31, 32, 351. X-5563 is a olasma cell neonlasm which arose in a female C3H mouse and which secretesyGz,-immunoglobulin [9, 33, 341. The above tumors were kindly made available to Dr Melvin Cohn by Dr M. Potter of the National Institutes of Health. All other tumors mentioned below were induced in BALB/c mice in this laboratory [4]. S63 [7] and S194 [42] are myelomas secreting IgA with anti-pneumococcal C-carbohydrate and anti-dinitrophenol antibody activity, respectively.

Lymphomas A small proportion of the tumors arising in oiltreated BALB/c mice are lymphomas [4], distinguishable from the myelomas by their cell morphology and failure to cause paraproteinemia in the host animal. Two such lymphomas were used for in vitro cultivation. The first, Sl, appeared in the peritoneal cavity of an ll-month old female BALB/c/St mouse injected intraperitoneally with T2, T5 and C bacteriophage in complete Freund’s adjuvant at 2 and 3 months, and with Bayol F at 3 and 5 months of age. The other, S49, was first transplanted from a 16 month-old female BALB/c/St mouse injected with phages at 3 and 4 months and with Bayol F at 4,6 and 8 months of age. Subcutaneous transplantation of each of these lymphomas yielded tumor growth mostly confined to the injection site but accompanied by enlargement of regional lymph nodes and the spleen of the host. Exptl Cell Res 60

Cultured cells were counted with a Coulter Counter Model F (Coulter Electronics, Hialeah, Flo.) after repeated aspiration in a pipette to disperse clumps and dilution into 0.9 % NaCl, 0.05 % formalin. Cell viability was estimated by the fluorescein dibutyrate (or diacetate) fluoro-chromasia technique [36] with dark field microscopy, using a Kodak Wratten No. 45 filter.

Karyo typing Mitotic

metaphase plate spreads of chromosomes

were obtained using a technique derived from me-

thods described by a number of authors. Cells were grown in the presence of 1O-6M colchicine for 3 or 4 h, washed, allowed to swell at 37°C in either 0.038 M sodium citrate for 15 min or diluted FEM (1:4 with water) for 20 min, sedimented and fixed by gradual addition of a fresh mixture of glacial acetic acidmethanol (1:3 by volume). The cells suspended in changed fresh fixative were then blaze-dried on cold wet slides [2].

Tumorigenic potential Tumor producing capacity of the cultured cells were tested by subcutaneous injection of IO6 to lo7 cells into host mice and subsequent examination for tumor growth at the injection site.

Cell stovage Tumor cells and tissue culture cells were susvended at 0°C in FEM containing 20% dimethylsulfoxide (DMSO). (Baker Chemical Co.) and 20 % fetal bovine ,&rum at a concentration of > 2 x lo6 cells/ml, slow-frozen in sealed ampoules in a Linde freezer and stored under liquid nitrogen [19]. When needed, am-

Mouse myelomas and ~ymp~omas iz culture pouies were thawed rapidly and the cells were suspended in FEM so that the concentration of DMSO was reduced to below 1 %.

lmmunoglob~lin production The ability of cells grown in culture to produce immunoglobulins was assayed by their ability to cause paraproteinemia in mice bearing tumors initiated by the cultured cells, or by their capacity to incorporate radioactive amino acids into material serologically identifiable as Ig. In the first method, serum from the tumor-bearing mouse was subjected to electrophoresis in 7.5 %-and 4.5 % acrylamide gels [8], the gels stained with amido black or Coomassie brilliant blue and the gammaglobulin region of the gel examined for the presence of bands which were much more heavily stained than comparable gels from sera of normal mice. In the second method, cells were grown in 3H-L-leucine containing FEM (minus leutine plus 10 % horse serum) for several hours, the cells removed by centrifugation and the culture fluid assayed for labeled secreted Ig by the direct or indirect serological precipitation method [39]. Direct method was used for MOPC21 and P3K labeliing experiment. In this experiment, secreted protein-[43]- was determined as follows. Cells were removed by centrifugation after labelling. Potassium phosphate-buffer (pH 7.2) was added to 0.1 M. An aliquot of the supernatant fluid was blotted on a piece of 2 cm2 Whatman No. 3 filter and washed four tnmes with cold TCA (5 % trichloracetic acid solution containing I % Bacto casaminoacids, Difco Laboratories, Detroit, Mich), once with hot TCA, once again with cold TCA and finally with ethanol. The samples were dried under infrared lamp and the radioactivities were determined in a liquifluor-toluene mixture (Nuclear Chicago Corporation, Des Plaines, Ill.). Ig synthesis was determined as follows. MOPC21 InG (diethvlaminoethvl-Sevhadex A50 fraction, vurifled according to the methods described [21])-was added to the above supernatant fluid at 200 pg/ml followed by a slow addition of Na,SO, (17 g/100 ml) with constant mixing at room temperature. After 1 h, the precipitate was collected by centrifugation, dissolved in-a small volume of 0.03 M potassium phosphate buffer at pH 8 and dialyzed against the same buffer at 4°C. Insoluble material after dialysis was removed bv centrifugation. To an aliauot of the dialysate, 0.06 O.D. unit (O.D. at 280 ‘x vol in ml) of MOPC21 IgG as a carrier and 20 ,ug of ovalbumin (Sigma Chemical Company, DeKalb Street, St. Louis, MO.) were added. At first, anti-ovalbumin (Hyland Laboratories) was added to the mixture, total volume adjusted to 0.5 ml with saline. incubated at 37% for I h and kept at 4°C for 18 h. The precipitate was centrifuged, washed 2 times with cold saline containing m-leucine, filtered through Millipore filter, and further washed on the filter which was dried and the radioactivity determined as above. A slight excess of anti-MOPC21 was added to the supernatant and the mixture was treated as above. The rabbit antiserum was prepared against purified MOPC21 serum immunogldbulin [?I] and was absorbed with normal

63

BALB/c serum. It reacted only with MOP621 serum or purified IgG from it as judged by imm~n~diffus~o~ test. The absorbed serum was concentrated with Na,SO, (antiMQPC21).

Agar cloning Minor modifications of the technique described. by Sanders & Burford [37] were used. Difco-Bacto agar was washed exhaustively with distilled water, and with acetone and dried under an infrared lamp. A nutrient agar underlay consisting of FEM, horse serum and either 0.6 or 0.75 % wasbed agar was prepared and 7 ml poured into each 60 mm Petri dish. Onto this hardened layer was then poured I ml of a molten (42°C) mixture containing FEM, 10 % horse serum and 0.2 or 0.37 % washed agar and the cells to be plated. During subsequent incubation at 37°C in 13 % CO, in air, the plates were examined for foci of cell growth.

RESULTS Initiation

of continuous cultures

The methods for somewhat from Since no attempt

culture initiation varied one cell line to another, has been made here to de-

fine a general method which would gaaranree successful culture of any lympboid tumor cell line, the life history of each esta will be described separately. 1. P3K: Cells from a MQPG21 tumor were cultured in FEM, plus 20 Y; horse serum for several months

during

which

time they grew very slowly with a populaiion doubling time of about 7 days. The cultured cells were stored frozen for several months after which tumors were reinitiated and maintained in

mice until culture was again attempted after nine further serial transplantations. Pieces of tumor were wetted with medium and printed onto plastic Petri dishes. The cells so released were incubated at a co~ce~tra~io~ pins 1096 of about IO6 cells per ml in FE horse serum with periodic additions of Lglutamine and folic acid to concentrations fter 4 of 100 and 0.8 pug/ml, respectively. days, the cells were transferred to fresh medium at a concentration of 1-2 x IO5 cells/ml. After 24 days during which glutamine and folic acid supplements and two partial

July 1967

Feb. 1967

April 1966

+ z!I

-

+’

40

50-l 12 widely distributed 41 and 70

80+2

+

&G) -

80+2

40+1

4Oil

60-253g

68+_3

70+2

68&2

+

+

-

-

&3 &3 -

72$2

m$’

none

1 LA

1 to 3 M 1to3s in various sizes and combinations Same as above lto6M

O-l M

0 to 2s none

g%;

1 M (med) 1 LA O-l M (med) No LA

1 M (med)

37 h (2 mos) 19 h (18 mos) 16h(2mos)

16 h (30 mos)

20-26 h (17 mos)

17 h (4 mos 20 % serum) 18-21 h (4 mos, 10 % serum) 18h(3mos)

36 h (4 mos) 25 h (8 mos)

20-22 h (17 mos)

21-25 h (20 mos)

16 h (25 mos)

Cell doubling timec

4.7

4.7

3.0

3.3

14

3.3

3.0

2.3

6.7

6.7

14

<4

<4

c4

14

c4

Drug sensitivityd TdR HCHS

4.6

5.8

4.5

4.2

8AG

a At various times of culture ranging from a few weeks to 3 years, tumor producing capacity of the cell line was tested according to the methods described in the text. b Marker chromosomes are designated as follows: M, metacentric; ,S’, subterminal; LA, large acrocentric; med, medium size, lg, large. Thus, 3 M (2 lg, 1 med) is read 3 metacentric marker chromosomes of which two are large and one medium in size. ’ The time in hours (h) required for a doubling of cell concentrations in the usual growth medium. As explained in the text, doubling time is affected by many factors. Different figures for one cell line indicate that in several experiments, the doubling time was found to be in this range. d Figures are drug concentrations (-log,, M) which just cause significant growth inhibition during a 4-day treatment/observation period. TdR, thymidine; HCHS, hydrocortisone sodium hemisuccinate; 8AG, 8-azaguanine. ’ Although XS63 originated in a mouse of BALB/c strain, this line now can grow in DBA/2/St as well as in BALB/c. ‘XC], a derivative of Cl, is capable of producing tumors in BALB/c mice, unlike its parental line, as well as in the original host C3H. Byo’typing was caout using a subli?ieoXiZh was+caured at 24” to 3rCfor more than two years. Colchicine treatment was done at 37°C. Chromo$om~numbers ;Sler&lus&redabou~ 65 h 70hand 630 to 140, Maker chron~sor$es v&re fgundjn va?iou&ombinations $ith mspeC;tto @zeagd type.

(BALB/c)

k/c)

g3H) X5563 (C3H)

Cl-18 (myeloma) xc1 (myeloma)

SlA (lymphoma) s49 (lymphoma)

Nov. 1966

May 1961

July 1968

+

June 1968

s194c2

+=

Jan. 1967

+

+

Mav 1967

June 1968

+

Feb. 1967

-

+

Turnorigenicity

‘Sept. 1966

Time initiated into culture

(BALB/c) X5563 (C3H)

(BALB/c)

ggw

ggw

m-2;)

Pl-17

MOPC 21 (BALE/c) Adj PC5 (BALB/c)

Origin (host mouse strain)

s194c5 (myeloma) Cl (myeloma)

5 5 P3K (myeloma) Pl-17 (myeloma) XPl-17 (myeloma) XPl (myeloma) XS63 (myeloma) s194c2 (myeloma)

&2 Cell line Q in culture g (cell type)

Immunoglobulin in host serum’ Chromosomes (type of Ig secreted) numbers markerb

Table 1. Properties of mouse myelomas and lymphomas in tissue culture

2 2a.

3

9 2. $ E R a

8

2

Mouse myelomas and lymphomas ifi culture Table 2. GompaCson of Ig synthesis by culture adapted and non-adapted myeloma cells

Cells MOPC21a 2.2 x 10’ (transplant no. 54) P3Kb 2.2 x lo7 ( > 2.5 years in culture)

protein’ (cpm)

Igd (%I

2.5 x lo6

32

2.1 x 106

40

a MOPC21 cells were obtained by mincing a tumor in FEM, lacking leucine, dissociating with a homogenizer [15], filtering through 200 mesh stainless steel screen and washing once with the medium. b Exponentially growing cells in culture were used after washing once with FEM minus leucine. ’ Labelling was carried out in 5 ml FEM minus leucine plus 10 % horse serum and 150,uCi 3H-L-leucine (spec. act. 750 mCi/mmole, Schwarz Bioresearch Inc., Orangeburg, N.Y.) for 20 h. d Direct precipitation method. Expressed as percentage of the secreted protein.

changes of complete medium were given, there was clear evidence of cell multiplication in the form of clusters of live cells. Such cells were gently dispersed by pipetting and diluted into fresh medium in which the population doubled every 2 days. Subsequent cultivation in FEM plus 10 % heated horse serum without supplementation resulted, within a few weeks, in a cell line with a doubling time of 16 h. It has maintained this growth rate (table 1) for over 3 years as stationary cultures in which most of the cells are detached from each other and from the surface of the culture dishes. Cells of P3K continue to synthesize Ig in vitro as well as in vivo (tables 1, 2). 2. H-17: At transplant generation number 65, cells from the Adj PC-5 myeloma were induced to form colonies in mouse spleens. One such colony yielded Pl-17 which karyotypically consisted of a single stem line unlike the uncloned tumor [43]. Cells from the enlarged spleen of a mouse bearing a 13th generation transplant of this clone were cultured as described for P3K for 19 days and 5 - 701812

65

then reestablished in mice as a tumor. After several unsuccessful culture attempts, the tumor line now having been through 12 further transplant generations was cultured again by the printing method with FEM further supplemented with pyridoxalQ.8, hydrocortisone 0.2, insulin 0.1, sodium pyruvate 22, L-serine 2. I, L-glutamine 117 pug/ml, horse serum IO Y; and non-essential amino acids (Microbiological Associates, Inc.). The starting cell density was about lo6 cells/ml. Weekly partial changes of medium were given and after 2 months, the cells could be successfully transferred into regular medium (FE heated horse serum and L-glutamine 117 yg/ ml) in which they have been grown ever since. 3. XPl-17: Some cells of the Pi-17 line after 66 days in culture were injected into a mouse and the resultant tumor used to reinitiate a culture line in regular medium which was maintained separate from Pl-17. After several months in culture, the cells were fou secrete no detectable Ig by the criteria of inability to cause paraproteinemia in host animals. uring the course of serial transpiantation of the myeloma Adj PC-5 a variant tumor line which secretes only immunoglobulin light chain was found to have replaced the original immunoglobulin secreting cells [4O]. This change was first noticed at transplant generation no. 80 and cells from generation no. 81 were used to initiate tissue cultures in FEM supplemented with pyridoxal, serine and insulin, as above, folic acid 8 ,ug/ml and 20 % fetal calf serum instead of horse serum. After 80 days, slow growth in this medium with transfers to fresh medium being made at 8- day intervals, the cells were grown in FEM with 20 “/b horse serum for 2 weeks and subsequently in FEM plus 10% horse serum. 5. XS63: A myeloma induced in this laboratory designated S63 has been found to Exptk CelERes 66

66 K. Horibata & A. W. Harris

secretean immunoglobulin with precipitating antibody activity against the C-carbohydrate of pneumococci [7]. Cells from transplant generation no. 5 were placed in FEM with 10% horse serum and within 2 weeks the cells were growing well. They were found not to secrete immunoglobulin detectable by acrylamide gel electrophoresis of serum from a tumor-bearing mouse [41]. 6. S194:The cells of this tumor line secrete an IgA with antibody activity [42]. Two culture lines were developed by alternately incubating tumor cells in FEM with 20 % horse serum for several days and then producing tumors in mice from the incubated cells for a total of two (S194C2) and five (S194C5)repeated cycles, respectively. They now grow in FEM with 10 % horse serum. 7. Cl: The continuous cell line was initiated from an X5563 tumor [9] in a C3H mouse (the strain of origin) at the 158th transplantation passageusing FEM with 10 % horse serum. 8. Cl-18: A spleencolony produced by cells of the Cl tissue culture line was established in culture as for Cl. 9. XCl: Some properties of this line have been described previously [41]. It arose in a culture initiated from transplant passagenumber 144 of the tumor X-5563 and supplanted the original population of IgG secreting cells. It does not synthesize detectable lg. 10. SlA: Cells were obtained from a small 17th transplant generation Sl tumor by manual mincing and removal of clumps by filtration through 200 mesh stainless steel grids and placed in FEM with 10 ‘$, horse serum at a concentration of lo6 cells/ml. Within a day or two, the population started to grow with a doubling time of 2 days and during subsequent passages into fresh medium over the course of several months, the doubling time gradually fell to a value at which it has remained for a year. Neither Exptl Cell Res 60

the original Sl tumor nor the SlA tissue cultured cells secrete detectable Ig. 11. S49: A tumor in the seventh transplant generation was dissociated with two strokes of a loose-fitting rubber-pestle homogenizer [15] and the cells in a 200 mesh grid filtrate were placed in FEM with 10 % horse serum at about IO6 cells/ml. During subsequent incubation, the cultures were monitored for viability and cell concentration and when the latter reached 1.5 to 2.0 x IO6 cells/ml, the cultures were diluted tenfold into fresh medium. Three weeks after the initiation of this line, the cells were growing rapidly at a rate which they have since maintained through hundreds of generations. They do not secrete detectable amounts of Ig. Properties of the cultured cells

Cell morphology: Growing cultures consist of spherical cells with the exception of the SlA line. Cells of this line are pleomorphic, consisting mostly of spherical cells but also of irregularly-shaped cells which exhibit slow ameboid movements even though they float freely in the medium and do not attach to the surface of the culture dish. Cooling of such cultures caused reversion to the spherical state. Stained smears of both strains of lymphoma cell showed the cells to be about 15,U in diameter with only a narrow region of cytoplasm surrounding each nucleus. The myeloma cells were typically much larger and contained relatively larger amounts of cytoplasm around their eccentric nuclei although the nuclear-cytoplasmic ratio differed considerably between different myeloma lines. Relative cell size distributions were obtained for several lines by counting the number of cells falling within successivethreshold intervals in the Coulter Counter (fig. 1). Since threshold values are proportional to particle volume, these distributions show that for a given ploidy level (for chromosome analyses,

Mouse myelomas and ~y~pho~as in culture

0

’

20

L 40

’ SO ’ 80

67

100

Fig. 1. Abscissa: threshold numbers; ordinate: relative cell number (%). S49, -•--; SlA (near diploid), -+-; Sl&4F;zetraploid), --A---; S194C5, not; Cell size istributions of some myeloma and lymphoma lines. Cells from exponentially growing cultures were counted repeatedly at successively larger threshold values in a Coulter Counter and the differences between counts for adjacent threshold values determined. The distributions were then normalized by taking percentages of the peak of each distribution.

see below), the myeloma cells are twice the volume of the lymphoma cells and that, for a given cell type, a doubling of chromosome number is associated with a doubling of cell volume. The counter was not accurately calibrated for measurement of actual cell volumes, but it was found that styrene divinylbenzene copolymer latex spheres of diameter 6-14 p (Bioproducts Division, Dow Chemical Co, Midland, Mich.) were mostly distributed between 2 and 15 threshold units. General growth characteristics: All of the above cell strains grow without attaching to the plastic substrate. The daughter cells generated by division adhere to each other loosely which results in the cultures being composed of single cells and clusters which are dispersible by repeated aspiration with a pipette without alteration of their growth rates. After initiation from solid tumors, the cuhures progressively grew faster until they stabilized with doubling times between 16 nd 26 h (fig. 2, table 1). They also gradually ecame better able to grow well at very low cell concentrations. Some lines, like XCl, P3K, and S49 are now able to grow with doubling times of 16 h at all concentrations

lO4b-

20

40

60

80

100

!20 --

!40

Fig. 2. Abscissa: time of incubation (h); or&afe: number of cells/ml. Quantitative growth of some myeloma cells in CUPture. Several replicate cultures of each cell line were initiated by diluting exponentially growing ceils into warm growth medium and grown under theconditions described in Materials and Methods. At inrervals, two cultures of each line were sampled and the cells counted in the Coulter Counter. The points on the growth curves are mean values of those pairs of cultures. The PI-17 cells bad previously been in culture for 20 months and XPl-17 for 17 months since culture initiation from the corresponding tumors.

Fig. 3. Electrophoresis of serum samples. 4.5 % po!yacrylamide gel, anode at the top. Normal C3H (1) CL initiated tumor-bearing C3H (2), X-5563 tumorbearing C3H (3), normal BALB/c (4), Adj PC-5 tumor-bearing BALB/c (5), H-17 initiated tumorbearing BALB/c (6), MOPC21 tumor-bearing (7), and P3K initiated tumor-bearing BALB/c (8). Exptl Cell Res 60

68 K. Horibata & A. W, Harris

Exptl

Cell Res 60

Mouse myelomas

and lymphomas

in dture

69

Fig. 4. (u-c) Metaphase chromosome pattern of XS63. Typical metaphase plate ccntaining 68 chrol (a), . __another representative ulate containing about 130 (b), and one containing extrer nely high number of some, approx.-250 (c). -

between 103 and lo6 cells/ml and can also grow when cultures are initiated from single clusters containing only about four cells. The latter observation was used to develop a technique for cloning of cell lines which could not be cloned by other methods. Cultures of all the cell lines reach a stationary growth phase at cell concentrations between lo6 and 2 x lo6 cells/ml. In this state, cultures of XC1 and XS63 remain viable for at least 2 days, but cultures of the lymphoma cells and of the Ig-producing myeloma cells deteriorate rapidly. Consequently, stock cultures of all cell lines are maintained by transfer of cells from exponentially growing cultures by dilution into fresh medium.

Cloning: Several thousand cells of each of several lines were plated in agar as described in Materials and Methods and for XC& XS63 and P3K colonies arose with efficiencies between 1 and 5 %. The plating efficiencies of SlA, 549 and S194 were tenfold or more lower. Well separated colonies could be removed from the agar with capillary pipettes and could be grown in the usual liquid medium. Most of the cell lines could also be cloned method mentioned by the “cluster-pick” above. In this method, about a t~o~s~~d cells were seeded into a dish of liquid fresh medium and allowed to grow for 2 days by which time the cells had multiplied to form

70 K. Horibata & A. W. Harris

Fig. 5. Typical metaphlase pattern chromosome of s194c2.

clusters of four to eight cells each. Isolated clusters were then located under the microscope, removed by capillary action into a drawn-out Pasteur pipette and deposited as microdrops into separate 30 mm dishes. After verification that each dish contained only a single cluster and no separated cells, 3 ml medium was added and the dishes incubated until the cultures had grown to a high cell concentration. Usually about half of the clusters picked in this way grew. Immunoglobulin synthesis: The myeloma lines Cl, Cl-18, Pl-17, P3K (table 2) and S194 synthesize and secreteimmunoglobulin (table 1) as shown by 3H-leucine labelling and serological precipitation [39] and by acrylamide gel electrophoretic analysis of sera from mice bearing tumors initiated by the cultured cells (fig. 3). Between 10 and 20 % of the total protein synthetic activity of the cells is devoted to Ig production. The lack of detectable synExptl Cell Res 60

thesis of Ig or its subunits by the mutant myeloma lines XC1 and XS63 has beendocumented thoroughly [41]. Preliminary experiments indicate that whereas XPI-17 secretes light chain at a rate 5 % of wild-type, XPl has lost this capacity entirely. Neither of the lymphoma lines secretes significant amounts of Ig (table 1) but there is preliminary evidence that they synthesize Ig which is not released from the cells in culture (Grant, G, Salk Institute, unpublished). Chromosome content: Metaphase plate preparations, obtained as described in Materials and Methods exhibited the features summarized in table 1 and some of them are shown in figs 4-8. Most of the myeloma lines are hypotetraploid as were the tumor cells from which the cultures were derived. Cloned lines of these cells showed karyological heterogeneity with a distribution of chromosome numbers around a mean number characteris-

6-2

tic of each line. Most also contained metacentric marker chromosomes (all 40 chromosomes in normal piploid mouse cells are acrocentric) which in some sublines of P3K increased in number during prolonged continuous cultivation. Two exceptional myeloma lines are XS63 which before cloning consisted of a roughly equal mixture of near tetraploid and near octaploid cell which coexisted in culture through at least 200 cell generations of growth (figs 4a, b, c, 9) and S194which shows a distribution of chromosome numbers around 40 (figs 5, 10). The latter displays some karyotypic instability and marker metacentrics have recently been observed in a

Fig. 6. (u-c). Representative metaphasa chromosome pattern of Cl (a), XC1 with 68 chromosomes (b) and XC1 with approx. 120 chromosomes (c). Arrows indicate subterminal marker chromosomes.

subline of S194C5.PI-17 contains one metacentric and one large acrocentric marker chromosome whereas XPl-17 no longer contains the latter. C1 and its derivative line, Cl-18 contain several subterminal marker chromosomes (fig. 6a). XC1 has lost these marker chromosomes (fig. 6b, c). One of the lymphomas, S49, has retained an apparently normal diploid number of acrocentric chromosomes through at least 500 cell generations in vitro (fig. 7). Only a very small proportion of S49 cells contain 39 or 41 chromosomes. The other lymphoma line, SIA, in the early stages of its growth in culture, was an apparently homogeneous po-

72 K. Horibata & A. W. Harris

Fig. 7. Typical metaphase chromosome pattern of S49. Fig. 8. Typical metaphase chromosome pattern of SlA subline. Arrow indicates a very long acrocentric “extra” chromosome.

pulation of cells containing 41 acrocentric chromosomes as was the tumor line from which it was derived. After several months continuous growth without cloning, it was seento comprise a mixture of cells with 40,41 and near 80 chromosomes, the near tetraploid cells progressively supplanting all others. While this line finally stabilized to form a population with a chromosome number distribution centered near 70, a subline consisting of cells containing almost exclusively 41 chromosomes was rescued from the mixed population by selection for resistance to the

growth inhibitory effects of thymidine (see below). The “extra” chromosome in this subline and in the earlier line is a very long acrocentric and is readily distinguishable from the other 40 (fig. 8). Tumor production: All of the cultured cell lines can initiate tumor growth in mice but some lines display peculiarities worthy of mention. While most of them can produce tumors following injection of lo5 and perhaps even smaller numbers of cells, the S49 lymphoma line after 2 years growth in vitro requires that at least lo7 cells be injected and

20

XS63

60

65

70

120

125

130

135

140

240

i

250

Abscissa: chromosome number per cell; ~&ate: % of cells analyzed. Fig. 9. Distribution of chromosome numbers in cells of XS63 line. Fifty metaphase plates were analyzed. Exptl Cell Res 60

i

Fig. 10. Abscissa: chromosome number per cell; ovdf-

nate: % of cells analyzed. Distribution of chromosome numbers in cells of S194C2 line. 100 metaphase plates were analyzed.

the resultant tumors grow much more slowly from which the culture was originally derived. No readily visible chromosomal change has accompanied this alteration. The Cl myeloma line produces tumors readily in C3H but not BALB/c mice, but the variant line XC1 as noted previously [41] is transplantable in both mouse strains. Furthermore, the S63 tumor originated in and is transplantable into BALB/c mice while its cultured Ig negative variant XS63 grows in DBA/2 and some other mouse strains but not in the BALB/c strain 1411.A BALB/c transplantable subline of XS63 has, however, been selected by passage in BALB/c mice treated with decreasing amounts of anti-lymphocytic serum (Cohn, M, Salk Institute, unpublished). Tumor-producing abilities of cultured cell lines are summarized in table 1. Response to thymidine: Growth of some representative myeloma and lymphoma cell lines in the presence and absence of various concentrations of thymidine (TdR) is shown in figs 11 u-d. Both lymphoma lines SlA and S49 are inhibited by concentrations above 10-5M than the lymphomas

15c)

0

50

100

‘50

Abscissa: time of incubation (h): or&unare: number of ceils/ml. (a-d). Titration of growth inhibiting activity of thymidine against myeloma and lymphoma cells. Ceils from exponentially growing cultures were diluted into fresh warm medium containing known concentrations of added thymidine. At intervals during subsequent incubation of these cultures, samples were counted in the Coulter Counter whose threshold was adjusted so that only cells within the normal size range were counted. 1, 2, 3,4, 5 and 6 indicate thynidine concentrations of 3 x IOF, 1QP, 3 x LO-4, IO-“, 3 x lo+, and 1O-5 M respectively. 0 indicates control with no addition of thymidine. Fig. II.

while the two myeloma lines are sensitive only to concentrations at least 30-fold higher than this. A cloned derivative of the neartetraploid line of SlA was found to be as sensitive to TdR as the near diploid line. The sensitivity of all the other myeloma lines falls within the range whose limits are defined by XC1 and P3K (table 1). Response to hydrocortisone: The concentrations of hydrocortisone sodium hemisuccinate required to cause cell growth inhibition were determined. The results (table 1) show that the lymphoma cells are about lOOO-fold more sensitive to the steroid than are the myeloma cells. One exception to this generalization was the myeloma line S194 which, during the

74 K. Horibata & A. W. Harris

early phases of its adaptation to tissue culture (2 months after initiation), was as sensitive as the lymphoma lines, but since that time, has repeatedly shown resistance comparable to that of the other myeloma lines, although no deliberate selection for resistance was applied in the meantime. Response to Gazaguanine: The sensitivities of some of the cell lines to the purine analogue 8-azaguanine are listed in table 1 as growth inhibitory endpoints assayed as the concentrations which allow the cells to grow through half the number of population doublings through which untreated cultures grow during a 4-day period. This test showed the SlA lymphoma line to be about 20 times more sensitive than the S49 lymphoma, the P3K and Cl myeloma cells. DISCUSSION The establishment of continuous tissue culture lines of cells derived from a number of mouse myelomas and lymphomas is described. Most of these lines have now undergone continuous growth in vitro for over 2 years which is equivalent to more than 500 cell generations. The procedures used to initiate these cultures varied somewhat from one tumor to another since they varied considerably in their ability to multiply when cell suspensions were simply incubated in the usual growth medium. For tumors which did not rapidly yield growing cultures, the technique of alternating incubation in vitro with growth as a tumor [3], has proved to be very useful. All of the cultured lines now grow well in the same medium. Most of the cell lines described here are karyologically aneuploid and somewhat unstable as are most other cells which have been grown in continuous culture by other workers. The exceptions are S194C2and S49 cell lines which have retained the normal number of Exptl

Cell Res 60

40 acrocentric chromosomes through many cell generations. In this respect and in some others, (steroid and thymidine sensitivity) the latter closely resembles the L5178Y murine lymphoblastic leukemia cell line of Fischer [lo, 16, 241. A number of the cell lines described in this paper have been found to continuously express the differentiated functions which are characteristic of their normal cells of origin. Some of the myeloma lines continue to synthesize and secrete homogeneous immunoglobulin (Ig) and one such line, P3K, has been employed by Schubert [39] to determine the pathway of Ig synthesis. The same cell line and the Cl, XC1 and S49 cells have been used by Kimmel [18] in a study of the synthesis and properties of species of RNA which are relevant to the myeloma cells’ production and secretion of Zg. His assays of cellular RNA, DNA and protein content quite closely parallel the relative chromosome numbers and cell sizes determined during the present work. Four of the lines described in this paper have partly or totally lost their Ig synthesizing capacity. The existence of these lines together with their “wild-type” parents, indicates the possibility for mutational analysis of the genetic control of Ig production. Currently, the main difficulty in this general approach to the genetic control problem is that we lack methods by which mutants can be deliberately induced or selected from Ig producing cell lines. An alternative method of genetic analysis involves the formation and isolation of hybrids between myelomas and other cell types. Unfortunately, the use of Sendai virus to form artificial hybrids l-13, 261 has not been successful since we have found that none of the Ig secreting myeloma cell lines (nor S49) are agglutinable by the virus, although the Ig negative mutant cells and the SIA lym-

phoma cells are agglutinable (unpublished experiments). Application of the drug resistant mutant cell hybrid selection system [22] is also difficult with the myeloma cell lines due to their near-tetraploid state and the consequent gene dosage of the thymidine kinase and the guanine-hypoxanthine phosphoribosyl transferase (PRTase) loci. The single near diploid myeloma line, S194, is so far the only exception to this difficulty and attempts are now beiag made to isolate kinaseless and transferaseless mutants of it. We have been successful in isolating both types of mutant by selection for resistance to 5bromodeoxyuridine and to 6-thioguanine or &-azaguanine respectively from both lymphoma cell lines (unpublished experiments). In preparation for attempts to select transferaselessmutant cells, several of the cell lines were screened for quantitative sensitivity to 8-azaguanine. One of the lymphoma lines, SlA, proved to be distinctly more sensitive than S49 and two myeloma lines. While this higher sensitivity may be just a peculiarity of that particular cell line, it is also possible that SIA and S49 represent neoplastic derivatives of different classes of lymphocytes which differ in this and perhaps other properties. Preliminary experiments by us and by others have demonstrated several other differences between the two lymphoma lines, such as the presence of a virus-like particle in S49 but not SlA; the possession of the TLI, 2, 3, antigens by S49 but not SlA (Hyman R, Salk Institute, personal communication), the difference in cell morphology and mobility described above and a difference in their response to hydrocortisone (Harris, A, in preparation). Thus, unless all of these differences are due either to the virus particles or to the single chromosome difference, the two cell lines may represent different physiological types of cell. The biochemical basis for the high azaguanine sensitivity is not yet clear.

We have found no difference in PRTase activity between SiA and S49 as judge quantitative enzyme assays. It has been known for several years that thymidine in high concentrations (about 1 mM) inhibits the multiplication of animal cells [48] and such treatment has been widely used as a cell synchronizing technique [I], However, cultures of both of our ~~rn~~~rna lines SlA and S49 are inhibited by thymidine concentrations down to 3 x lop5 M. The myeloma cells are relatively resistant and in this respect behave like most other types of cultured cell. Sensitivity to thymidine appears, then, to indicate a characteristic metabolic difference between Iymphomas and m~e~o~as and, therefore, perhaps, between lymphocytes and plasma cells. Similar degreesof sensitivity have been reported for cultured ly~~~~oblas~i~ cells of both murine [24] and human [38] origin. Furthermore, there is evidence that thymidine has a quantitatively similar inhibitory effect on normal lymphocytes [47j although evidence on plasma cells so far is lacking. Further work is presently being done with the lymphoma cells to determine the biochemical basis for high Td its genetic control through the isoiation of TdR-resistant mutants. The ameboid movement displayed by SlA cells in suspension is another activity in which this cell line at least behaves like normal lymphocytes, and cells of this line eornmonly appear in the ‘“hand-mirror” farm described for normal lymphocytes [45]. The other lymphoma line, S49 has never been observed to execute this type of rnove~e~t~ A property which both lymphoma ceils share with normal lymphocytes is sensitivity to the cytotoxic action of hydrocortisone. The continuous cell lines provide a conveaient system in which to study the mode of killing of lymphocytes by steroi Harris, A W, Tomkins, G

76 K. Horibata h A. W. Harris preparation). Further experiments on steroid sensitivity have been carried out and will be reported soon (Harris, A. In preparation). It has been stated that normal plasma cells, unlike lymphocytes, are completely unaffected by corticosteroids [45]. All of the myeloma lines except one are highly resistant to inhibition by hydrocortisone. The exceptional myeloma line, S194, appeared to be sensitive very early in its life history, but subsequent tests have shown it to be resistant. Thus, while steroid resistance seemsto be a natural property of myeloma cells, the possibility that these cells acquire resistance during transplantation or growth in culture has to be entertained. Selection for the resistant state in culture seemsunlikely, since we have found (unpublished experiments) that there is no significant selection pressure for resistance of the lymphoma cells when they are grown for long periods of time in the usual medium. Resistant lymphoma cells emerge only when deliberate selection with added steroid is applied. The failure of steroids to be useful as therapeutic agents in human multiple myelomatosis in contrast to their dramatic effects on lymphoblastic leukemia suggests that myeloma cells are naturally resistant. If, as some evidence suggests [12, 14, 451, lymphocytes can differentiate into plasma cells, steroid sensitivity and thymidine sensitivity are functions which must be altered during that transition. We wish to thank Drs L. Sachs, K. Bhargava, R. Hyman and D. Schubert for their participation in various aspects of this work. We also wish to thank Ruth Eostein for her excellent technical assistance. This investigation was supported by Public Health Service Research Grant, no. CA-10311-02 from the National Cancer Institute, and Damon Runyon Memorial Fund no. 942,942A. 942B to Kengo Horibata: a Damon Runyon Memorial Fund Research Fellow: ship, no. DRF-400 to Alan W. Harris; and by Public Health Service Research Grant. no A105875 from the National Institute of Allergy and Infectious Diseases, and a Public Health Service Training Grant, no. CA0521301 from the National Cancer Institute, both to Dr Melvin Cohn. Exptl

Cell Res 60

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Received October 2, 1969