Urinary proteins inhibit fibroblast proliferation

(;en. phormm. Vol. 10. pp. 153 to 155 C Pergamon Prc~,s Lid 197t1 Printed m Great Brllalfl

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URINARY PROTEINS INHIBIT FIBROBLAST PROLIFERATION GABRIEL HOCMAN. ANTON B6ZNER. VLADIMIR LACKOVI(~. IVAN Ci~N,~R and HANA HO~TACKA Research Institute of Gerontology. 90101 Malacky, Czechoslovakia

(Received 12 June 1978) Abstracr---I. Peptides from normal human urine (molecular weights above 500; 1000 and 10,000daltons) inhibit the proliferation of human fibroblast cells in cultures. 2. This inhibition appears both in the presence and absence of calf serum in cultures and is dosedependent. 3. The fraction of proteins having molecular weights above 10,000 daltons is the most potent inhibitor but seems to be non-toxic for the cells.

having molecular weights higher than approximately 500daltons; UM-2 for approx. 1000daltons and UM-10 for approx. 10,000 daltons. Pooled human urine (I 500 ml) (females. approx. 30 years old) were divided into three times 500 ml and each portion was concentrated on a different membrane. Thus, from the same urine, three freezedried samples of urinary proteins were obtained : one, consisting of compounds of molecular weights higher than approx. 500 (labeled UP 500); second, consisting of compounds having molecular weights higher than 1000 (UP 1000); and third, consisting of compounds with molecular weights higher than approx. 10,000daltons (UP 10,000).

H u m a n blood serum as well as homogenates of tissues contain a n u m b e r of c o m p o u n d s which influence the growth and mitotic activity of various cells (Carpenter & Cohen, 1976; Michl, 1977). They may stimulate (Rinderknecht & Humbel, 1976; M a r e c et al.. 1977) or inhibit (Jansa & Kolfif, 1977) their proliferation. These c o m p o u n d s seems to be mainly various peptides or l o w - - m o l e c u l a r weight proteins. Urine is considered to be a renal filtrate of blood, into which, in general, c o m p o u n d s having molecular weights lower than that of the a l b u m i n (approx. 67,000 daltons) pass almost freely. We may, therefore, assume that the mentioned stimulatory or inhibitory c o m p o u n d s will be found in urine, too (Carpenter & Cohen, 1976). The aim of the present c o m m u n i c a t i o n is to examine the influence of isolated c o m p o u n d s from normal h u m a n urine, having molecular weights higher than approx. 500 daltons, upon the growth of h u m a n fibroblast cells in cultures.

The fibroblast cultures Normal human diploid fibroblast cells (from embryonic lung) obtained in 19th passage were cultured in Eagle's basal medium (BEM) containing antibiotics, with a certain amount of urinary proteins (see Results) and with--or without--10% inactivated (56°C, 30 min) calf serum (ICS) at 3T~C for 72 hr, till a monolayer was formed. The initial number of cells was 5 x 105 in each sample, in 5 ml of the medium. After the incubation, the cells were released from the monolayer by 0.25% trypsin solution and counted.

MATERIALS AND METHODS

Urinary protein samples

Isolation of urinary compounds Normal human urine was sterilized as soon as possible (hr) after obtaining it by passing it through a bacterial filter, and stored frozen (-20°C) without the addition of any preservatives till the processing (days-weeks). The thawed urine (500ml) was subjected to ultrafiltration (Diaflo, Amicon, Holland) and concentrated to approx. 50-70ml. The concentrated sample contained the higher molecular weight compounds such as peptides, polysaccharides etc. For reasons of simplicity, we will refer to compounds in this concentrated sample as to "'urinary proteins". To the sample of urinary proteins in the ultrafiltration apparatus. 100-150ml of 0.9% sodium chloride solution was added and the ultrafiltration repeated until the volume of the sample was diminished again to 50-70 ml. Then the addition of sodium chloride solution and ultrafiltration was repeated in the manner described above (in all, 2 or 3 times). The last sample or urinary proteins, obtained after the described washing, was devoid of most low-molecular weight organic compounds and its salts were substituted by sodium chloride. This sample was freeze-dried. In the process of ultrafiltration, three types of membranes were used: U M ~ 5 for the retention of compounds

Each of the three freeze-dried urinary protein samples (UP 500; UP 1000; UP 10,000) was dissolved in 10ml of distilled water. Since none of the samples dissolved completely, the obtained suspension was centrifuged (Janetzki K 23 centrifuge, 10ml, 5 rain, 3000rpm, approx. 1000g) and only the clear supernatant was used in the experiment. We assumed that most proteins and peptides were in the solution while the gross amount of polysaccharides formed the precipitate. In the clear supernatant the protein content was determined according to the standard Lowry's procedure. The obtained protein concentrations for each sample were: UP 500, 1.23 mg/ml; UP 1000, 1.89 mg/ml; UP 10,000, 0.61 mg/ml. From these solutions of urinary proteins certain amounts were pipetted into the incubation medium (sec Tables). (BPS means buffered 0.9,% sodium chloride solution, used to make up the required volume of the sample). RESULTS AND DISCUSSION The composition of each sample, the final n u m b e r of cells and the percentage of increase of the n u m b e r of cells at the end of the" incubation period are presented in Tables 1, 2 and 3, representing the addition of three different samples of urinary proteins.

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154

GABRII!I. HOCMAN el a].

Table I. Influence of UP 500 upon the proliferation of human fibroblasts Cell suspension

BEM

ICS

BPS

UP 500

UP 5(FO (mg/

(ml)

[ml)

(mll

(ml)

(ml)

sample)

2.5 2.5 2.5 2.5 2.5

1.8 1.8 2.5 2.25 2.0

0.2 0.2 0 0

0.5 0 0 0

0

0

0 0.5 0 0.25 0.50

0 11.61 0 0.30 0.61

Number of cells in

Number of cells

sample

(°,,)

12 6 5 4.5 2.5

x l0 s x 105

100 50

x 10 s

lO0

x IOs x 10"~

90 50

Table 2. Influence of UP 1000 upon the proliferation of human fibroblasts Cell suspension (mlt

BEM (ml)

ICS (ml)

BPS (mll

UP 1000 tml)

UP 1000 (mg/ sample)

2.5 2.5 2.5 2.5 2.5

1.8 1.8 2.5 2.25 2.0

0.2

0.5 0 0 0 0

0 0.5 0 0.25 0.50

0 0.85 0 0.42 0.85

0.2

t) 0 0

Number of cells in sample 12 7.5 5 5 3.5

Number of cells (";,)

x l0 s x IOs

100 62.5 100 100 70

x IOs x 105 x 105

Table 3. Influence of UP 10,000 upon the proliferation of human fibroblasts Cell suspension (ml)

BEM (ml)

ICS {mll

BPS (ml)

UP 10.000 (ml)

UP 10,000 (mg/ sample)

2.5 2.5 2.5 2.5 2.5

1.8 1.8 2.5 2.25 2.0

0.2 0.2 0 0 0

0.5 0 0 0 0

0 0.5 0 0.25 0.50

0 0.30 0 0.15 0.30

The results presented in the tables offer a n u m b e r of various conclusions. There is approximately the same a m o u n t of lower molecular weight peptides (between approx. 500 a n d 10,000dahons) as higher molecular weight peptides and proteins (above 10,000 daltons) in urine. By proteins and peptides we mean in this respect the compounds in the freeze-dried urinary samples giving positive reaction in Lowry's standard procedure for determination of proteins. In the sample U P 10.000 we found approx. 6 mg of soluble proteins a n d peptides in 500 ml of urine. This value, which should be ragarded as rather approximative, refers to the soluble proteins having molecular weights higher than 10,000 daltons. In both samples U P 500 and U P 1000 were found approx. 12- 18 mg of soluble peptides and proteins in 500 ml of urine. This value, again, approximative, refers to all peptidic c o m p o u n d s having a molecular weight above 500 or 1000dahons. Since no signs of deterioration of cell cultures were observed, we assume that the preparations of urinary proteins obtained by means of the described procedure are nontoxic, devoid of organic and inorganic substances from the urine which may be toxic to h u m a n fibroblast cells. The presence of urinary proteins inhibits the proliferation of fibroblast cells both in the presence of calf serum and in its absence, too. T h a t seems to indicate that the urinary proteins contain an inhibitor of fibroblast growth. That this inhibition is not caused

Number of cells in sample 12 5 5 2 I

× x × × x

10s 10"~ l0 b IOs l0 s

Number of cells ("',3 100 41.5 100 40 20

by a mere absence of a stimulating, or perhaps, a nutrient factor, is indicated by the fact that even in the presence of ICS and other necessary factors the urinary proteins slowed down the proliferation rate of fibroblasts. In the presence of calf serum as well as in its absence, the most intense inhibition of fibroblast growth is caused by the urinary protein fraction containing c o m p o u n d s of molecular weights higher than approx. 10,000daltons. However, both other samples of urinary proteins exhibit a certain inhibitory activity and in all three samples is this inhibitory effect dosedependent: the higher the concentration of a given urinary protein fraction in the sample, the more intense the inhibitory effect. All three urinary protein samples, however, exhibited inhibitory effects differing in their intensity. It could, therefore, not be excluded that the urinary proteins consist of a c o m b i n a t i o n of stimulating and inhibiting factors, having different molecular weights and, hence appearing in various relative concentrations in all three samples of examined urinary proteins.

ttypothetical considerations We assume that the blood serum of n o r m a l adult individuals contains factors that stimulate the proliferation of fibroblasts as well as factors which inhibit them. There is, perhaps, quite a n u m b e r of both factors in the serum. The stimulating factor, or. rather, a complex of

155

Urinary proteins inhibit fibroblast proliferation various stimulating peptides and proteins, isolated by Michl (1977) who called it pleiotropin, exhibits a higher molecular weight and therefore is less readily excreted by the kidneys into the urine. The inhibiting factor(s) are probably peptides having a much lower molecular weight and therefore are readily excreted into the urine. It could not be excluded that between these two controversially acting factors there is a kind of equilibrium. Perhaps one molecule of the stimulating factor could be split to produce two inhibitory factors, or vice versa. We assume that in blood of young individuals the stimulating factors prevail, causing a quicker healing of wounds, and, perhaps, other effects, characteristic for young age, too. On the other hand, in blood of old persons prevail the inhibitory factors, as one of the characteristic features of the old age. It has been proven that blood serum from old individuals, used instead of calf serum in the media for culturing fibroblasts causes a much slower proliferation of cells as sera from young persons (Hocman, unpublished results). These inhibitory factors, excreted into the urine of

old persons should cause a more intense inhibition of fibroblast proliferation as the similar urinary proteins isolated from the urine of children or adults. To p r o v e - - o r - - r e j e c t - - t h i s hypothesis, work is in progress in our laboratory.

REFERENCES

CARPENTERG. & CO, EN S. (1976) '251-labeled human epidermal growth factor. J. Cell. Biol. 71, 159-171. J^NSA P. & KOL~I~ A. (1977) Chalony: tklifiov6 specifick~ inhibitory mitozy. Bioloyick~ L/sty 42, 266--274. MAREC J., KEPRTOV./~ J., SPURN.A V., MINAI~OV./L E. & MICHL J. (1977) Effects of the growth-promoting alpha globulin/GPAG/on the in vitro incorporation of exogenous DNA into mammalian cells. Experientia. 33, 33-34. MICHL J. (1977) Bun~nd Kultury, p. 158. Academia, Praha. RINDERKNECHTE. & HUMBELR. E. (1976) Amino-terminal sequences of two polypeptides from human serum with non-supressible insulin-like and cell-growth promoting activities. Evidence for structural homology with insulin B chain. Proc. ham. Acad. Sci., U.S.A. 73, 4379-4381.