CA1291055C - Method and cell line for obtaining plasminogen activators - Google Patents

Abstract

ABSTRACT

A chemically defined growth medium designated as SYC medium, containing for example fetuin, bovine serum albumin, insulin, transferrin, 5.alpha.-dihydrotestosterone and dexamethasone, is suitable for adapting a plasminogen activator-secreting cell line to growth in the absence of fetal calf serum.
Such cell lines are capable of producing relatively large quantities of plasminogen activator.

Description

- BACKGROUND OF:THE INVENTION

Plasminogen actlvator (PAj is a serine protease wh:ich~ ex~erts its action throuqh hydrolysis of the ~
Arg56~0~ Va1561 peptide:~bond~in plasminogen, yielding : 5 the 2-chain pIasmin molecùle.~ Plasmin has a general proteolytic activity and in the~physiologica.l ~, .
environment of circulating blood it attacks mainly fibrln. ;~Plasminogen activator~plays a key role in the fibrinolytic system, which~by lysing intra- or :extra~-vascular thrombi, clots, or fibrinous deposits, : profoundly influen~ces the incidence of throm~oembolic : ; :: vascular disease anfl 1ts~outcome.
.~2~ plAsminogen activators have been isolated~from body fluids such as blood:and:urine, and solid ~`3~ ~ 15 tissues of various histo~aic origin. ~Mammallan cell cult~res are also known to produce plasminogen activators. There may be large differences in the ~; amou~ts of plasminogen acti~ator produced by cell lines derived from different tissues of the same ,~
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animal, by cells of the same histoloqic type derived from cells of a single type independently transformed with the same oncogenic agent.
There is an urgent need for obtaining cells wh-ch are capable of producing increased amounts of PA and establishing techniques which would improve the process of using animal cell culture to produce ~ PA on an industrial scale~
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- 10 European Patent Application 113,319 A2 discloses human cell lines capable of proliferating in the absence of serum and other macromolecular growth factors as well as the method utilized for producing such cell lines. European Patent Application 15 112,174 A2 describes a serum-free medium capable of growing a wide range of suspension and monolayer cells, said medium containing fetuin, transferrin and a substituted phosphatidyl-choline.
As taught in J. Biol. Chem., 256, 7035-7041 ~ 20 (1981), it i5 well-known that human cell lines of - neoplastic origins, e.g. melanoma cells, can produce substantial amounts of PA.
Malignant cell lines such as cervix carcinoma, larnyx carcinoma, epidermoid carcinoma of the oral 25 cavit~, colon and rectum carcinoma ~ells have been shown to produce PA (see Molecular Cellular Bio-chemistry, Vol. 15, No. 2, pages 149-153 (1977).
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In Proc Natl. Acad. Scl., Vol. 7a ~ No. 9, pages 5673-5676 (1981), it is reported that two metastatic prostatic carcinoma cell lines, PC-3 and DU-145, have been grown in long-term culture in a defined medium free of serum, hormones or growth factors.
A progressive loss of metastatic potential and tumorigenicity was noted following serial passage in vitro of human epidermoid carcinoma. However, with regard to PA, high levels of production were 10 well-correlated with retention of tumorigenicity and metastasis, Cancer Research, 40, 2310-2315 (1980).
Barnes and Sato in "Growth of a Human Mammary Tumour Cell Line in a Serum-Free Medium" (Nature, October 1979) report the growth of the MCF-7 cell 15 line in a serum-free medium supplemented with insulin, transferrin, epidermal growth factor, prostagladin F2 and cold-insoluble globulin. The synthetic nutrient medium used in this study was 1:1 mixture of Ham's F-12 and Dulbecco modified Eagle's ; 20 medium supplemented with antibiotics, sodium bicarbonate, HEPES and sodium selenite.
None of these references suggest or teach that the yield of PA from PA-secreting cells can be in-creased by adapting such cells to grow in a medium 25 which is esse~tially free of fetal calf serum.

S-JMMARY OF THE INVENTION

The present invention is directed to a method of increasing the yield of plasminogen activator (PA) , .
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obtained from a PA-producing cell line, and a cell line capable of producing relatively large amounts of PA. The method involves growing PA-producing cells in a suitable growth medium containing fetal calf serum (bovine serum) and adapting the cells to grow in said suitable growth medium essentially free of fetal calf serum by passing the cells through a series of said growth media containing decreasing amounts of fetal calf serum.
A biologically pure tissue culture, capable of ; producing relatively large quanti~ies of plasminogen activator is a human prostate adenocarcinoma, availa-ble from the American Type Culture Collection (ATCC) designated as Cell Repository Line (CRL)~1435. Other 15 biologically pure tissue cultures capable of producing relativel~ large quantities of plasminogen activator when adapted according to the method of the present invention are ATCC CRL-1622 and ATCC
CRL-1579.

DETAILED DESCRIPTION OF THE INVENTION

It is well-known that plasminogen activators can be produced by tumor cells in culture. Examples of tumor cells from mammals which can be used in the present invention include melanoma, pro~tate, breast, 25 colon, ovarian, pancreatic, cervical, rectal, endometrial and fibroblastlc tumor cell lines.
Preferred cell lines are human carcinoma cell lines such as melanoma, prostate, breast, colon, ovarian, :'.

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pancreatic and endometrial. Suitable carcinoma cell lines are available from depositories such as American Type Culture Collection, 1201 Parklawn Drive, Rockville, MD 20852 or can be obtained from numerous researchers at universities, hospitals or research institutes. The identity of the carcinoma cell line is not of critical importance; the present ; method allows the amount of PA obtained from a PA-producing cell line to be increased by as much as 10 from about 5 to about 60 fold. As indicated herein, a preferred high-yield producer of PA is a human prostate adenocarcinoma, ATCC CRL-1~35. Other preferred biologically pure tissue cultures capable of producing relatively large quantities of 15 plasminogen activator when adapted according to the method of the present invention are ATCC CRL-16~ and ~- ATCC CRL-1579.
; As used herein, the phrase "suitable growth medium" refers to a medium such as described in 20 detail hereinafter containing a mixture of Waymouth's MB752/1, Dulbecco Minimal Essential Medium (MEM) and Ham's F-12 madium in a ratio (by weight) of from about 1:1:1 to about 3~1:2, respectively.
~he method involves selecting a paren~ carcinoma 25 cell line which produces PA, an~ growing the cell line to confluency in a suitable growt~ medium which contalns at the outset from about 5 to about 20 percent fetal calf serum. After the cell li~e reaches confluency, the cells are removed, usually by 30 trypsinization and subjected to subculturing i~ a series of suitable growth media which contain ~IS-1338-CIP

decreaslng amounts of fetal calf serum. The rate of removal of the fetal calf serum can be easily determined by sequentially reducing the calf serum and examining the viability of the cell growth. For example, the parent cells can be grown in a suitable growth medium containing approximately 10 percent fetal calf serum to confluency. The cells are then removed and subcultured through at least one passage in a suitable growth medium containing approximately 10 5 percent fetal calf serum. After the cells reach confluency, the cells can be removed again and subcultured through at least one passage in a suitable growth medium containing approximately 2.5 percent fetal calf serum. This procedure can be ; 15 repeated until the suitable growth medium used is essentially free of fetal calf serum. In the present inventionl sequential reduction by a 2-fold factor ; has been found to be a preerred method.
It has been found that various combinations of 20 commercially available growth media are suitable for use in the present invention. However, as the concentration of fetal calf serum is decreased, the suitable growth medium used in the method of the present invention must be correspondingly 25 supplemented with the following growth factors:
fetuin, bovine serum albumin, insulin, transerrin, 5~-dihydrotestosterone and dexamethasont. The point dar;ng the sequential reduction of fetal calf serum at which these growth factors must be added may be 30 readily determined by one skilled in the art and will vary depending on factors such as the type of cell ~ ~*()~5 lines being cultured and the specific conditions under which the culturing is being carried out.
Suffice it to say that the growth factors must be added at a point when cell viability miyht otherwise be threatened due to the decreased concentration of fetal calf serum; however, when the suitable culture medium contains a sufficient concentration of fetal calf serum to sustain the growth of the cell line, addition of the growth factors noted above is 10 unnecessary and may lead to su~sequent difficulty in adapting the cell line to grow in the suitable serum-free growth medium.
A suitable growth medium for use as described herein can be obtained by mixing together Waymouth's 15 MB752/1 medium, Dulbecco MEM and Ham's F-12 medium, commercially available from GIBCO Laboratories,Grand - Island, New York. The ratio (by weight) can range from l:l:l to 3:1:2, respectively.
A preferred medium, hereinafter referred to as 20 "SYC" medium, is composed of a 1:1:1 (by weight) mi~ture of Waymouth's MB752/1, Dulbecco MEM, and ~am's F-12, respectively. It has been observed that cell growth is optimized when the suitable yrowth medium used is SYC medium.
In the total absence of ~qaymouth's MB752/1, cell growth is greatly retarded. These media have the compositions as set forth in Table I.

` -TABLE I
~Concentration of each component in mg/l) Waymouth Ham's Dulbecco SYC
MB752/1 F-12 MEM Medium CaC12 (anhydrous) 90.61 33.22 200 107.94 CuS04 5H20 __ 0.00249 -- 0.00083 Fe(N03)3 9H20 0.1 0.033 FeS04 7H20 __ 0.834 -- 0.278 KCl 150 223.6 400 257.87 2P04 80 ~~ ~~ 26.67 MgC12 112.56 57.22 -- 56.59 MgS04 97.67 -- 97.67 65.11 NaCl 6000 7599 6400 6666.33 Na2HP04 300 142.04 -- 147.35 NaH2Po4 H2o 125 41.67 ZnS04 7H20 __ 0.863 -- 0.288 L-Alanine -- 8.9 -- 2.97 L-Arginine HCl75 211 84 123.33 L-Asparagine H20 -- 15.01 -- 5.0 L-Aspartic acid 60 13.30 -- 24.43 L-Cystine~2HC119.55 -- 62.57 27.37 L~Cysteine HCl-H20 100.26 35.12 --- 45.13 L-Glutamic acid 150 14.7 -- 54.9 L-Glutamine 350 145 584 360 L-Glycine 50 7.5 30 29.17 L-Histidine HCl H20 164.1 20.96 42 75.69 L-Isoleucine 25 3.94 105 44.65 L-Leucine 50 13.10 105 56.03 L-Lysine HCl 240 36.5 146 140.83 L-Methionine 50 4.48 30 28.16 L-Pheny]alanine 50 4.96 66 40.32 L-Proline 50 34.5 -- 28.17 L-Serine -- 10.5 42 17.50 ., ~.
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MB752/1 F-12 MEM Medium L-Threonine 75 11.9 95 60.63 L-Tryptophane 40 2.04 16 19.35 L-Tyrosine (Na salt) 57.66 7.78 103.79 56.41 L-Valine 65 11.70 94 56.9 Ascorbic acid 17.5 -- -- 5.83 Biotin 0.02 0.0073 -- 0.0091 Ca pantothenate 1.0 0.48 4.0 1.83 Choline Cl 250 13.96 4.0 89.32 Folic acid 0.4 1.30 4.0 1.9 i-Inositol 1.0 18.0 7.2 8.73 Nicotinamide 1.0 -- 4.0 1.67 Niacinamide -- 0.037 -- 0.012 Pyridoxine HCl 1.0 0.062 4.0 1.69 Riboflavin 1.0 0.038 0.40 0.48 Thiamine HCl 10.0 0.34 4.0 4.78 Vit. B12 0.2 1.36 -- 0.52 D-Glucose 5000 1802 1000 2600 Glutathione (reduced) 15 -- -- 5 Hypoxanthine (Na salt) 29 4.77 -- 11.26 Phenol red 10 1.2 15 8.73 Thymidine -- 0.73 -- 0.24 Linoleic acid -- 0.084 -- 0.028 Lipoic acid -- 0.21 -- 0.07 Putrescine 2HCl -- 0.161 -- 0.054 Na pyruvate -- 110 110 73.33 :

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o As described herein, as the concentration of fetal calf serum is serially decreased in each pass-age, it becomes necessary to supplement the suitable growth medium with from about 50-150 m~/l fetuin;
from about ~0-150 mg/l bovine serum albumin; from about 5-10 mg/1 insulin; from about 5-40 mg/l transferrin; from about 50-200 ~gil ~ 5-dihydrotestosterone;~and from about 50- 00 ~g/l - ~; dexamethasone. It has been observed that insulin and 10 transferrin are crltical growth factors without which the cells will die~after one passage where all growth factors would otherwlse have been added to suppIement ;; the~suitable growth medium. `
The following examples are pxovided as a means 15 of illustrating the present in~rention and should not be construed as a limitation thereon , ~ ~ Example I
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Following receipt from the depository, a human prostate adenocarcinoma, ATCC CRL-1435, was stabil-20 ized by growing to confluency in Earle's Minimal ; Essential Medium ~M~M) commercially available from GIBCO. This procedure was carried out by placing a 25 ml portion of MEM plus 10 percent f~tal calf serum ~ in a 175 ml culture flask, maintained at 37C in a 5 ; 25 percent CO2 atmosphere, and adding 5 x 10 of the above adenocarcinoma cells. The parent cells were removed~ from the flask by adding a 5 ml portion of 0.25 percent trypsin.

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The parent cells from the above stabilization procedure were then added to a previously prepared uantity of SYC medium containing 10 percenk fetal calf serum and were maintained at 37C. The cells reached confluency in approximately one week after ~` which they were removed~by trypsinization as described above and were added to a quantity of SYC
~ medium containing 5 percent fetal calf serum. The `~ cells were again maintained at 37C until they 10 reached confluency. This procedure was repeated twice using SYC medium containing 2.5 percent and 1.25 percent fetal calf serum, respectively. At the 1.25~percent fetal calf serum concentration, the SYC
medium was supplemented with fetuin (75 mg/l), bovine ; 15 serum albumin (75 mg/l), lnsulin (8 mg/l), transferrin (25 mg/l), 5~-dihydrotestosterone (100 ~g/l) and dexamethasone (100 ~g/l). The cells reached confluency in approximately ten da~s.
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The cells were then passaged into SYC medium 20 containing the same growth factors a.s in the 1.25 percent fetal calf serum passage, except that no fetal calf serum was present. The cells reached con~luency in approximatel~ one week.
The above cells were then passaged ten times 25 through SYC medium (containing the same arowth factors as above), free of fetal calf s rum, until ; the cell line stabilized. Determination of .: ~
~f stabilization was achieved by observing the same growth pattern during each passage and the amount of 30 PA produced. After ten passages, the biologically ~`- pure altered confluent cells, designated PC-3f, and , -,,, : ,,. ~ ,, :
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~,.2s~n~s ~ 12 -deposited with ATCC having accession number CRL-8539 were assayed for PA production by the following procedure (a.s described in Progress in Chemical ~- Fibrinolysis and Thrombolysis, Vol. 3, pages 315-322;
1978).
A 100 ~l portion of culture fluid (obtained ~-after confluency) was incubated with 100 ~l of human plasminogen (0.33 mg/ml) for 15 minutes at 37C after which 250 ~1 of Tris buffer (0.1 M Tris; 0.2 M ~aCl;
10 pH 7.4) and 250 ~l of lmM tripeptide substrate (Val-leu-lys-p-nitro-anilide; Kabi, Stockholm, Sweden) ` were added. This mixture was incubated ~or an additional 3 minutes after which the reaction was stopped by the addition of 100 ~l of 50~ acetic acid.
15 The absorbance of the reaction mixture was read at 405 nm and was then compared to a urokinase standard curve~generated with commercially available urokinase (Calbiochem., La Jolla, CA) using the same assay procedure. This assay technique allows quantitation 20 of the PA produced by the cells by measuring the ~.
quantity of plasmin produced by activation of the plasminogen from the PA present in the culture fluid.
The test results are set forth in ~able II. The amount of PA produced by the cells i5 expressed in 25 CTA units per ml of culture fluid as stipulated by ~ the Committee on Thrombolytic Agents of the National - ~eart Institute, as described ln Throm~. Diath.
.aemor., 21, page 259 (1969j. As a control, the PA
activity of the parent cell line (~TCC~1435) was also 30 determined.

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~ TABLE II
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~:: : Cell Line 48 Hours 72 Hours 96 Hours ATCC CRL-1435 :11.5 18.5 Died :
PC-3f (ATCC CRL-~
: 8539) 28.5 45.0 70.4 . As~shown in:the above test results, passage of : the parent cell line through serum-free medium pro-duced a cell line which yielded up to 70.4 CTA Units 10 of PA, as compared to a maximum of 18.5 CTA Units of , :~ PA produced by the parent cell line, an increase of : .
:~ approximately 4-fold. Advantageously, the altered . .
~: cell: line was viable for an additional 24 hours.
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Examp~e II

. 15 The procedure described in Example I was repeated using as a parent cell line a human pancreatic carcinoma cell line, ATCC-1420. The : biologically pure altered cells obtained by the ~ procedures described herein were desisnated as :::: 20 PaCa-2f and were deposited w-th ATCC having accession number ATCC ChL-3725. Test results obtained are summarized in Table:III below:

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TABLE III .
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(PA activity expressed in CTA Units/ml) Cell Line 48 Hours 72 Hours 96 Hours -~

.~ ATCC-1420 2.5 7.5 Died PaCa-2f~ATCC CRL-~
:: 8725) 5.4 15.0 25.2 As shown in the test results, passage of the parent cell llne through~serum-free medium produced a :~ cell line which yielded PA up to 25.2 CTA Units, an 10 increase of approximately 3-fold, as well as a 24 ~ :`
~ hour increase in viability.
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The procedure described in Example I was ~: repeated using as a parent cell line a mouse melanoma ~`: . 15 cell line, B-16, available from ~ackson Lab., Bar ::' Harbor, Maine. The biologically pure altered cells obtained by the procedure described herein were : designated as B-16f. Test results obtained are summarized in Table IV below:
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~ ' : ~PA activity e~pressed in CTA Units/ml) Cell Line 24 Hours 48 Hours 72 Hours 96 Hours B-16 0.70 1.20 Died ---~ .
~ 5B-16f 2.30 %.80 3.0 3.2 i~
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~: The biologically pure altered cell line showed a : 3-fold increase in PA production at 24 hours and a ` 2-fold increase in PA production at 48 hours.
-; Furth:er/ the altered cell line was viable for up to 10 96 hours.
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Example IV

.{ The procedure described in Example I was . repeated using as a parent cell line ATCC CRL-1622, an endo~etrial adenocarcinoma cell line available 15 from the ATCC. The biologically pure altered cells obtained by the method:of the present invention were designate~ as KLEf and were deposited with ATCC
~ having ~ccession number ATCC CR~-8726. PA activity : - : was measured at 48 hours after the cells reached ;~ Z0 confluency and the results obtained are shown in ~ Table V.
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TABLE V
~ ~, (PA activity expressed in CTA Units/ml) Cell Llne _ _ 48 Hours ATCC CRL-~1622 0.05 ~: 5 KLEf (ATCC CRL-8726) 3.03 ~' ' ;~ As shown in Table V, the altered cell line,; KLEf, showed over a 60-fold increase in PA production '~ over the:parent cell line at 48 hours.

Example V

The procedure described in Example I was repeated~using as a parent cell line an amelano~ic : melanoma cell line available from ATCC under ' accession number ATCC CRL-1579. The biologically ~, pure altered cells obtained by the method of the 15 present invention were designated MLf and were deposited with ATCC having accession number ATCC CRL~
, 8774. PA activity was measured at 48 hours after :"~ the cells reached conflu~,ncy and the results obtained ; are shown in Table VI.

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ML (ATCC CRL-87~4) 0.07 ~ f Actlvity level was below detectlon llmits of the :~: assay.

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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are de-fined as follows:

1. A chemically defined growth medium desig-nated as SYC medium wherein SYC medium is composed of a 1:1:1 (by weight) mixture of Waymouth's MB752/1, Dulbecco MEM, and Ham's F-12, respectively.

2. The chemically defined growth medium of claim l containing fetuin, bovine serum albumin, insulin, transferrin, 5.alpha.-dihydrotestosterone and dexamethasone.

3. The chemically defined growth medium of claim 2 containing fetuin in a concentration of from 50-150 milligrams per liter; bovine serum albumin in a concentration of from 50-150 milligrams per liter;
insulin in a concentration of from 5-10 milligrams per liter; transferrin in a concentration of from 5-40 milligrams per liter; 5.alpha.-dihydrotestosterone in a concentration of from 50-200 micrograms per liter;
and dexamethasone in a concentration of from 50-200 micrograms per liter.

4. The chemically defined growth medium of claim 3 containing fetuin in a concentration of 75 milligrams per liter; bovine serum albumin in a concentration of 75 milligrams per liter; insulin in a concentration of 8 milligrams per liter; transfer-rin in a concentration of 25 milligrams per liter;
5.alpha.-dihydrotestosterone in a concentration of 100 mic-rograms per liter; and dexamethasone in a concentra-tion of 100 micrograms per liter.