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Cell Research 121 (1979) 277-282

Experimental

DIFFERENTIAL

METABOLISM

CHIN.ESE

HAMSTER

OF MANNOSE CELL

BY

LINES

ROLF H. DAHL and M. L. MORSE Webb-Waring University

Lung

Institute

of Colorado

and the Department Medical

Center,

of Biophysics Denver,

and Genetics,

CO 80262,

USA

SUMMARY In contrast with the CHO-KI Chinese hamster cell line, the V79 cell line was found not to grow on the sugar mannose, and in fact to be inhibited and killed. Study showed no differences between the lines with regard to mamrose transport, mannose specific energy metabolism, or effect of mannose on energy or nucleic acid metabolism. The V79 line lacked an a-mannosidase which the CHO-KI line possessed.

In a study to ascertain the carbohydrates upon which Chinese hamster cells could grow [ 11 it was found that only one of the hamster cell lines was able to grow on mannose. The tissue source of the strains was embryonic lung (V79) and the ovary (CHOKl). Only the ovarian strain grew on mannose. Since the earlier study we have extended our studies in order to discover the reason for the failure of the lung cells to grow. A portion of the results are presented here.

MATERIALS

(FCM) prepared by passing FCS over a Sephadex G50 column [6]. FCM was added to 10% of the total volume. The saline solution used for washing and other manipulation of the cells was G-saline [6] with glucase omitted. This is essentially a phosphate-buffered saline containing calcium and magnesium. The cell culture condition for the following measurements for enzyme activities, CO, production, and

AND METHODS

Cell lines and culture conditions Two Chinese hamster cell lines were studied. CHOKl was isolated from the hamster ovary [2], and V79 from fetal luna r3i. Routine m&t&ance of these cultures in F12 medium supplemented with 8% fetal calf serum (FCS) (F12FC8) was as described previously [4,5]. To test the ability of these cell lines to grow on various carbohydrates, the sugars in question were substituted for the ghtcose of F12 at an equal concentration [l]. In these cases the serum supplement was the macromolecular portion of fetal calf serum

1. Abscissa: time (min); ordinate: [14C]mannose cpm X102/7X 104cells. [W]Mannose uptake by hamster cells. Cl, V79 cells; 0, CHO-Kl cells.

Fig.

Exp CellRes121(1979)

278

Dahl and Morse Table 2. Conversion of uniformly labelled mannose to labelled CO2 Strain

cpm/h/lW cells

Incubation in G-saline CHO-KI v79

3 330 3 564

Incubation in F12 with 5 % FCM CHO-Kl 1290 v79 2 780 Hybrid between V79 and CHO-KI 1 670

Fig. 2. Abscissa: mannose cont. (mM); ordinate: rel. plating efficiency. Relative plating efficiency of CHO-KI in various mixtures of mannose and glucose. Glucose concentrations: 0, 10; 0, 1; l ,0.4; q ,O.l; * ,0.05 n&i.

uptake of sugars was to inoculate 5X 105cells/60 mm diameter plastic Petri dishes. After 24 h the cultures were dense but not confluent with many mitotic cells present. At this time the cultures were prepared as indicated for the following assays.

Enzyme assays For the measurement of enzyme activities the cells were harvested with trypsin, centrifuged and washed twice with an equal volume of G-saline without glu-

Table 1. Some enzymatic activities of Chinese hamster cells nm NADPHl minlmg protein

Activity

Strain

Mannokinase

CHO-KI v79 CHO-Kl (1)

0.87 1.06 970

v79 CHO-Kl v79 CHG-Kl v79 CHO-Kl v79

%E 595 31.2 57.0 6.93 8.39 2.08 2.05

Phosphomannose isomerase Hexosephosphate isomerase Glucose&phosphate dehydrogenase Glucokinase

Exp Cell Res lZt(1979)

case, and resuspended at lx lo7 cells/ml in 0.01 M NaPG4 buffer, pH 7.2. The cells were lysed by three cycles of freeze-thawing and the lysate was used directly for the enzyme assays. The ability of cell extracts to hydrolyse p-nitrophenyl derivatives of various carbohydrates was determined by adding an amount of extract to give 0.1 mg protein to 1 ml of reaction mixture containing 0.1 M acetic acid-sodium acetate, pH 4.5 and 0.01 M p-nitrophenyl derivative, and incubating at 37°C for 15 h. The reaction was terminated by the addition of 2 ml of 0.23 M Na&OJ, the solution clarified by centritirgation, and the absorbance at 415 run measured. Assay of the enzymes involved in glucose and mannose metabolism was based on the methods of Beutler & Tepple [7] in which the rate of the coupled reduction of NADP is measured at 3443nm. These assays were carried out by adding an amount of lysate to equal 5X1@ cells to a reaction mixture of 1 ml containing 0.1 M Tris HCl, pH 8.0; 2 mM h&Cl,; 0.2 mM NADP; and the additional components listed below for each particular activity assayed. The assay for mannokinase included 2 mM neutralized ATP; 2 mM mannose; 0.1 U glucose-6phosphate dehydrogenase (G6PD); 0.1 U phosphoglncose isomerase (PGI); and 1.56~10-~ U phosphomannose isomerase (PM). The phosphomannose isomerase assay included 2 mM mannose-&phosphate; 0.1 U G6PD; and 0.1 U PCI. The hexosephosphate isomerase assay included 2 mM fructose&-phosphate and 0.1 U G6PD. The G6PD assay included 2.0 mM glucose and 2 mM neutralized ATP.

CO, production The production of CO* by cultures inoculated and grown in F12FC8 as indicated above was determined by washing the plates twice with 5 ml G-saline without glucose, addmg 2.5 ml of G-saline without gtucose or F12 (adjusted to pH 7.2 with HCl) witbontglucose plus 5% PCM, and adding 2.5 &i UL [W&nannose. The plates were placed in a closed container at 3PC and continuously lluahed with a stream of air which gently bubbIad thmqh 1 ml of 17% KOH. At 0 and 60 mitt, 0.1 ml of the KOH solution was removed,

Mannose metabolism

in hamster cells

279

cant differences between the two lines in duction from uniformly labelled glucose by the steps of mannose metabolism leading to the formation of glucose. Mannose is phosCHO-Kl and V79 cells phorylated by mannokinase and then con14C09cpm/lOB cells/h verted to glucose phosphate by an isomerGlucose Glucose+mannose ase [lo]. As is shown in table 1, there appears to be no significant differences in the CHO-Kl 9 950 15 050 levels of these enzymes in the two strains. 14 ooo 13 200 v79 There are no differences also in the early Sugar cont.: 0.2 mM for ducose and 10 mM for mansteps of glucose metabolism, and these ennose. Incubation was in G-saline. zymes were not found to be inhibited by mannose (unpublished observation). added to 8 ml of a scintillation solution containing 2 Since metabolic steps subsequent to gluparts 0.8% 2.5 diphenyl oxazole (PPG) in toluene: 1 part Triton X-100 and counted. Under these conditions cose phosphate are in the glucose pathway 14Ccounting was 90 % efficient. it was not thought, since V79 grows on glucose, that any of these later metabolic steps Transport studies could be affected. However, it is possible The uptake of radioactive carbohydrates was measthat one of these later steps might be manured by the method of Platter & Martin [8]. This technique consists of inoculating cells (number of cells and nose sensitive. A check on this could be growth was as above) to Petri dishes containing covmade in two ways: measurement of carbon erslips. The coverslips were rinsed twice with G-saline without glucose, incubated in G-saline without glucose dioxide production from uniformly labelled containing the tested radioactive sugar at 1.0 pCi/ml, mannose; measurement pf carbon dioxide and incubated at 37°C. At various times the coverslips were removed, rinsed rapidly by dipping sequentially production from uniformly labelled glucose in three reservoirs containing 125 ml G-saline without in the presence of unlabelled mannose. As glucose, dried, and placed in a scintillation vial for determining by liquid scintillation counting the radiois shown in tables 2 and 3, there is no efactivity retained. This uptake activity was destroyed fect on carbon dioxide production from gluby heating to 85°C for 5 min. cose by mannose, and there is no signifCell hybridization cant difference between strains with regard Sendai virus cell fusion was performed as described to carbon dioxide evolution from mannose. Table 3. The effect of mannose on CO,pro-

by Kao et al. [9].

RESULTS It was speculated initially that some simple difference might account for the failure of the V79 strain to grow on mannose, such as a transport defect, or a deficiency of one of the enzymes specific to mannose metabolism. This was not found to be the case. Fig. 1 shows the uptake of [14C]mannose by each strain and that, in fact, V79 appears to transport mannose more rapidly than CHO-Kl. In the other vein, there were no signifi-

Table 4. Complementation analysis by hybridization of mannose sensitive and insensitive cell lines Colonies/total no. of cells plated” Cells hybridized

Expt 1

Expt 2

v79 x v79 V79x CHO-gly A V79x CHO-glyZC1 CHO-gly Ax CHO-glyA

012.8x 105 24/2.7x 105 -

0/4.4x 105 92/4.2x 1W 0/4.0x 1W

a Cells were fused with aid of inactivated Sendai virus and plated directly to F12~FcM10 with 10 mM mannose and no glucose at a density of 0.5-2x 104cells/ 60 mm plate. Exp Cell Rbs 121 f/979)

280

Dahl and Morse 2.

1. 9 .8 .7 .d 3 A

.3

.3

2 .\

.I

1

2

3

4

5

I9

Fig. 3. Abscissa: mannose COW. (mM); ordinate: rel. plating efficiency. Relative plating efficiency (HO cells/colony) of hamster strain V79 in various mixtures of mannose and glucose. Glucose concentrations: 0, 10; 0, 1.0; l ,0.5; 0,O.i; * ,0.05 mM.

Fig. 4. Abscissa: time (days); ordinate: 110.of colonies with greater than 50 cells/colony X fff. The effect of glucose and mannose on the survival of V79. Two hundred V79 cells were inoculated to F12 medium containing 10% FCM but lacking glucose, with the following addition- of glucose or mannose or glucose plus mannose. At the indicated times the medium-was aspirated and F-12 medium plus 10% FCS containing 0.01 M glucose was added. The plates were stained and counted after 9 days’ total incubation. The results show that attachment rate, and extent of attachment, is independent of carbohydrate, and that mannose not only does not supportgrowth, but is toxic to V79 cells. Carbohydrate present at inoculation: 0, 5X10m4glucose; 0, no carbohydrate; t, lop2 mannose; q ,lO’* M mannose+5x 10” M glucose.

The above observations indicate that the difference(s) between strain CHO-KI and strain V79 was not a defect in the pathway of energy metabolism. It is possible, by the cell-fusion procedure [9], to obtain information about the nature of the differences between the CHO-Kl and the V79 cell lines. In this procedure cells are fused by employing inactivated Sendai virus, and then asking if the fused cell, now a near-tetraploid, is able to grow on mannose. In this way it is possible to see if the two cells entering into the fusion can complement each other. From the result it is possible to infer something about the nature of difference between the two cell lines

fused; e.g., whether the difference is dominant .or recessive. Cells of V79 were fused with CHO-Kl and the resultant near-tetraploids placed in medium containing mannose as energy source. Fusion products grew (table 4), indicating complementation and that the inability of V79togrow behaved as a recessive. (J-HO-Kl- was therefore supplying some function that was missing in V79. The-natureof the deficiency in V79 could be a step-in nucleic acid synthesis sensitive to,mannose. This possibility was studied by measuring the labelhng of cellsx with radioactive thymidine in the presence and absence of mannose. The results showed that

Exp Cell Res 121(1979)

Mannose metabolism in hamster cells Table 5. Mannosidase activity of Chinese hamster cell lines Line

run p-Nitrophenyl-a-mannoside hydrolysed per hour

CHO-Kl v79

5.15 0.17

mannose does not appear to interfere in cellular labelling with thymidine whether measured by uptake or when the labelling was measured by autoradiography (data not shown). The above observations show no defects in energy metabolism or in the synthesis of nuclear material. Carbohydrates are also incorporated into cellular material via transferases, forming a number of complex polysaccharides and glycoproteins. It has been known for some time that some genetic diseases result from a failure of cells to degrade some of these complex substances, leading to what are called ‘storage’ diseases [ 111. Such storage diseases are often the result of a single enzyme defect such as a galactosidase or a mannosidase. Cells derived from individuals with storage diseases, when placed in culture, are sensitive to the carbohydrate which they cannot metabolize properly. The results from some earlier growth experiments had suggested that the V79 strain might be sensitive to mannose in some unspecified manner. Therefore, a study of the effect of mannose on the growth of the CHO-Kl and V79 strains was initiated. These studies were made by following the effect of varying concentrations of mannose on the ability of the cells to grow on varying concentrations of glucose. The results are obtained as plating efficiency at different ratios of mannose to glucose in the

281

medium. The results from these studies are shown in figs 2 and 3, where it appears that mannose has essentially no effect on the growth of the CHO-Kl, but seriously inhibits growth of the V79 strain. The action of mannose on V79 cells is more than inhibiting, since cells placed in the presence of mannose lose their plating efficiency more rapidly than in the absence of any carbon source (fig. 4). Because of the above results, V79 and CHO-Kl cells were tested for cr-mannosidase activity. The results are given in table 5 and clearly show that CHO-Kl is able to hydrolyse p-nitrophenyl-cr-mannoside, whereas V79 is not. Thus, a defect in polysaccharide degradation may be the basis for the difference in mannose metabolism of the two cell lines. DISCUSSION It was not expected that tissues derived from an animal would differ in metabolism of a simple substance such as mannose. Organs may of course vary in their metabolism but usually only for the case of special substances metabolized or catabolized in small amounts. Thus, the observations that ovary strain CHO-Kl possessed an enzyme not found in the line derived from embryonic lung was surprising. These strains did not originate from a single animal and each has had an independent laboratory history and the reason(s) for the difference(s) remains obscure. The observations on the V79 line indicate that in fact this cell line is sensitive to mannose. Whether the sensitivity is owing only to a a-mannosidase deficiency is not known, nor is it known whether the sensitivity is correctable by supplementation with external a-mannosidase. A preliminary experiment employing externally Exp Cell Res 121 (1979)

282

Dahl and Morse

REFERENCES added jack-bean a-mannosidase suggests Dahl, R H, Morrissey, A, Puck, T T & Morse, M not, but the question of a-mannosidase L, Proc sot exp biol med 153 (1976) 251. specificity has not been investigated. 2. Puck, T T, The mammalian cell as a microorganism. Academic Press, San Francisco (1972). V79 derivatives that are no longer sensi3. Chu, E H Y, Sun, N C & Chang, C C, Proc natl tive to mannose have been observed, but acad sci US 69 (1972) 3451. 4. Ham, R G, Proc natl acad sci US 53 (1%5) 288. the nature of the mechanisms underlying 5. Ham, R G & Puck, T T, Methods in enzymology the loss of sensitivity remain to be eluci(ed S P Colowick & M P Kaplan) vol. 5, p. 90. Academic Press, New York (1%2). dated. 6. Kao, F-T & Puck, T T, Methods in cell physiology The observation that CHO-Kl has an en(ed D M Prescott) vol.-& p. 23. Academic Press, New York (1974). zyme lacking in the other strains provides 7. Beutler, E &Yepple, L, J clin invest 48 (1969)461. a biochemical marker which characterizes it 8. Platter, H & Martin, G M, Proc sot exp biol med 123 (1966) 140. and the information should be useful in the 9. Kao. F-T. Johnson. R & Puck. T T. Science 164 future for the identification of this cell line. (1969) 357. ’ The deficiency in V79 provides a cell line 10. Stein, M W, J biol them 1% (1950) 753. 11. Meersmann, G, von Figura, K & Buddecke, E, useful in confirming the location of the huHoppe-Seyler’s Z physiol them. In press. man genes for the cu-mannosidases that hu- Received0ctober17 1978 Revised version received December 27, 1978 mans possess. Accepted January 26, 1979

Exp Cell Res I21 (1979)