Detachment variants of Chinese hamster cells

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Experimental Cell Research I I9 (1979) 327-332

DETACHMENT Hyaluronic

VARIANTS

OF CHINESE

Acid as a Modulator

B. J. BARNHART,’

HAMSTER

CELLS

of Cell Detachment

S. H. COX and P. M. KRAEMER

Cellular and Molecular Biology Group, Los Alamos Scientific Laboratory, University of California, Los Alamos, NM 87545, USA

SUMMARY Variants of the Chinese hamster cell line CHO have been isolated and characterized with respect to attachment and trypsin- or EGTA-mediated detachment kinetics, cell morphologies, and the complex carbohydrates (labeled with [SH]glucosamine) of the cell surface. The variant which was more readily detached from the substratum exhibited a more rounded cell shape and had three times more label as hyaluronic acid on the cell surface than the parental cell. The slowly detaching variant had a morphology similar to the parental cell but only half the radioactivity ascribable to hyaluronic acid. Endogenous levels of CAMP were unaltered in the variants. Exogenous dbCAMP caused the cells to elongate and flatten but did not alter the characteristic detachment kinetics. The role of hyaluronic acid as a modulator of the cell substratum interface is discussed.

Alterations in cell-surface biochemistry have been reported to coincide with variants in detachability from the substratum. Pouyssegur & Pastan isolated variants of BALB/c 3T3 cells which were more sensitive than normal cells to detachment reagents [l] and which were later shown to be deficient in glycosylated membrane proteins [2]. We have previously reported that a variant of the cell line CHO which was more resistant than normal to detachment with trypsin and EDTA had no detectable hyaluronic acid on the cell surface [3]. To investigate further the possible role of glycosaminoglycans at the cell periphery, we have isolated and cloned a collection of detachment variants of the cell line CHO, all of which display deviations from parental cells in detachment from the growth substratum. The altered levels of hyaluronic acid on these cells are discussed relative to the hypothesis previously put forth from

this Laboratory [3] that this complex carbohydrate acts as a modulator of the accessibility of attachment sites to detaching agents. MATERIALS

AND METHODS

Selection of detachment variants Chinese hamster cells, line CHO [4], were grown in Ham’s F-10 medium [5], supplemented with fetal calf serum (FCS), (lo%), penicillin (100 U/ml), andstreptomycin (100 pg/ml) in humidified incubators with 5 % carbon dioxide. Cells were mutagenized as monolayers of 2X 109cells/75 cm* at 37°C for 24 h with 200 pg/ml of ethylmethane sulfonate. This procedure vielded about 60% survival. The cells were washed free of the mutagen and incubated for 7 days before selection. To enrich for detachment variants, the monolayer of mutagenized cells (1 x 108cells/75 cm*) was exuosed to 5 ml of 50 &ml trvosin (crvstallized three times, Calbiochem) hr~‘Puck’s’ saline-b [6] for 5 min at 3?C. The Bask was shaken for 5 set at 120 reciprocals/mm on an Eberbach shaker. To obtain rapidly detaching variants, the detached cells were then inoculated into fresh medium and grown to near confluency. To obtain slowly detaching variants, the 1 To whom reprint requests should be sent. Exp Cell Res 119 (1979)

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Cox and Kraemer

Fig. 1. Abscissa: time (min); ordinate: fraction detached. Kinetics of detachment from plastic substratum. Cells were inoculated into T25 flasks at 3 x lo5 cells/ flask and incubated overnight at 37°C. Prior to the detachment assay, the cells were washed once with Pucks saline-G and shaken on a reciprocal shaker to remove loosely attached cells. Trypsin in saline-G was added and, after the designated incubation periods, a separate flask was shaken to detach loosened cells. Additional trypsin was added to each flask to detach remaining cells. Details are given in the Materials and Methods section.

cells remaining attached to the flask after 10 min of the above trypsin treatment and shaking were grown to near confluency. These respective enrichment procedures were sequentially repeated 10 times. Clones from single-cell isolates were obtained and checked for the desired detachment characteristics with trypsin. Monthly checks for contamination with MycoDlasma using the House & Waddel modification [71-f ihe agar plate method of Chanock et al. [8] have been negative for the parental CHO cells and all variants in our collection. The variants were stored at -85°C.

Detachment

and attachment

assays

To measure detachment kinetics, cells were inoculated into T25 flasks at 3x 105cells/flask and incubated overnight (16 h) at 37°C. This inoculum provided a sufhcient number of cells for counting and minimized cellcell contacts. Prior to the detachment assay, the cells were washed once with 5 ml of prewarmed saline-G. While in the wash, the cells were shaken for 5 set on the reciprocal shaker (120 reciprocals/min) to remove loosely attached cells. The wash was decanted, and 5 ml of warm trypsin or the calcium chelating agent ethvlenenlvcol-&is-(B-amino-ethvl ether)-ZV.iV’-tetraaceiic ahd (EGTA) at 100 p&ml in saline-G were added. After designated times at 37°C. one flask of each variant and one of the parental cells was shaken for 5 set, as described above, and detached cells were decanted into chilled tubes. Trypsin (5 ml) was added to each flask to detach the remaining cells, which were then decanted into chilled tubes. All cells were counted in an electronic particle counter. Attachment rates were measured using cells which were either harvested from suspension culture or from monolayer by trypsinization. Cells were inoculated Exp CellResl19(/979)

time (min); ordinate: fraction attached. O-O, Parental CHO cells; O-O, slowly detaching variant DV2; A-A, rapidly detaching variant DVll. Kinetics of attachment to a plastic substratum. Cells were harvested from monolayer cultures by trypsinmediated detachment, the trypsin was neutralized with an equal volume of serum-containing medium, the cells were disnersed bv ninettinz. and cell counts were obtained using an electronic particle counter. Cells were inoculated into T25 flasks at 3x105 cells/flask and. after designated incubation periods, a separate flask of each clone and of the parental cells was shaken for 5 set (see Materials and Methods) on a reciprocal shaker, and the unattached cells were counted. Attached cells were trypsinized and counted. Details are given in the Materials and Methods section.

Fig. 2. Abscissa:

into T25 flasks at 3x105 cells/flask and, after designated times at 37°C one flask of each clone and of the parental cells was shaken for 5 set, as described above, and the unattached cells were decanted into chilled tubes. The attached cells were removed with trypsin, and all cells were then counted in an electronic particle counter. All detachment and attachment assays were performed in a 37°C Envirco warm room.

Complex carbohydrate

determinations

The labeling, harvesting, and fractionation of the complex carbohydrates have been described previously [3, 91.

RESULTS The rationale that the enrichment procedure outlined in the Materials and Methods section would provide variants with enhanced or diminished ease of detachment to the substratum proved to be correct. The trypsin-mediated detachment rates of DV 11 and DV 12 are greater than the parental line, and the rates of DV2 and DV3 are less (fig.

Detachment variants of Chinese hamster cells

Fig. 3. Cell morphologies in the presence and absence

of db-CAMP. Approx. 3 x 105cells were inoculated into 60 mm plastic Petri dishes and incubated at 37°C. After 3 h, 1 mM db-CAMP was added to one dish of parental cells and to one of the rapidly detaching variants, DVl 1, and incubation was continued for 24 h. Phase contrast photomicrography was performed on viable

329

cell cultures at x260. To include a larger number of cells in the fields, especially dense areas of the cultures were selected for these photographs. (a) Parental CHO cells; (b) parental CHO cells+1 mM dbCAMP; (c) DVll cells; and (d) DVI 1 cells+1 mM db-CAMP.

Exp Cd/ Res 119 (1979)

330

Barnhart,

Cox and Kraemer

600

600

100 0

0

IO

20

Fig. 4. Abscissa:

f;q~;). O-O,

30

40

50

60

70

fraction no.; ordinate: cpm of 3H label; A-A,

80

PO

radioactivity cpm of %

DEAE-cellulose separation of trypsin-removable material from CHO cells and detachment variants. Each profile represents 10’ cells that had doubled in cell number during incubation in labeling medium containing [3H]glucosamine and Na2?30,. GP, glycopeptides; HA, hyaluronic acid; HS, heparan sulfate; and CS, chondroitin sulfate.

1). The difference in detachment between DVl 1 and the parental line can be seen also when the culture flasks are vigorously shaken by hand in the absence of trypsin or EGTA. Reciprocal agitation, which removes only 5% of the parental cells, deExp Cell Res 119 (1979)

taches 50 % of the variant culture. The relative kinetics of detachment for DV2 and DVl 1 with EGTA were similar to the trypsin detachment data. Although the variants displayed different detachment rates, the rates of attachment to the substratum were not significantly altered from normal (fig. 2). The data shown are for reattachment of trypsinized monolayer cells, but similar results were obtained for attachment of cells grown in suspension culture. Thus, while detachment and attachment rate assays permit characterization of cell-substratum interaction, these parameters should be viewed as indicators of either biochemically distinct aspects of that interaction or biochemically similar but different quantitatively. When grown in a subconfluent monolayer, parental CHO cells exhibited a somewhat flattened polar configuration (fig. 3 a). The morphology of DV2 and other slowly detaching variants is like that of the parental cells [3]. The rapidly detaching variants such as DVl 1 are somewhat more rounded and epithelial-like in shape, as shown in fig. 3 c, and are suggestive of a modified attachment. When DVll was grown in 1 mM dibutyryl adenosine 3’ : 5’-cyclic monophosphate (db-CAMP) for 24 h at 37”C, the cells became flattened and elongated (fig. 3 d) as do DV2 and the parental cells shown in fig. 3 b. However, the detachment kinetics remained the same as variant cells cultured in the absence of the cyclic nucleotide. This indicates that cell morphology is not necessarily related to detachability. That detachability and cell morphology are not correlated with endogenous CAMP levels is suggested by our findings that the parental cell and cell detachment variants we have tested have approx. 10 pmols of cAMP/106 cells. These estimations were performed as previously described [3]. There was no ap-

Detachment

Table 1. Comparison of radioactivity ascribable ment variants and parental CHO cells ;YctpmllO’cells) KZlO’ Hyaluronic

variants of Chinese hamster cells to the complex carbohydrates

cells)

331

of detach-

DV2 (cpm/lO’ cells)

acid

TRM 4 3H Pellet:’ 3H

II0

1 737 (289)b 549 (499)

7 548 6 772

7 246 (96) 8 059 (119)

7 246 (96) 7 043 (104)

2 372 4997 0.75 0.76

2 348 (99) 4997(100) 0.59 (79) 0.70 (92)

2 728 (115) 4 697 (94) 0.79 (105) 0.80 (105)

586 842 0.97 0.90

486 (83) 850 (101) 0.69 (71) 0.86 (96)

527 (90) 699 (83) 0.94 (97) 0.92 (102)

23 828 7 548 II 107 2 872

26 2ll(llO) 6 265 (83) II 773 (106) 1 897 (66)

23 351 (98) 7 397 (98) 10 774 (97) 2 901 (101)

601

337 (56) 78 (71)

Glycopeptides

TRM, 3H Pellet, 3H Heparan

TRM, Pellet, TRM, Pellet,

sulfate

3H 3H 3”S/3H “S/3H

Chondroitin

sulfate

TRM, 3H Pellet, ‘H TRM, 35S/3H Pellet, “WH Total radioactivity

incorporated

[3H]glucosamine 35S-inorganic sulfate 3H as TRM 35Sas TRM

a TRM is defined as the trypsin-removable material [9]. b Figures in parentheses are percentages of the parental cell values. c Pellet refers to the sedimented and resuspended cells following trypsinization to remove TRM.

parent effect of cell density on either cell morphologies or detachment kinetics. We investigated the cell-surface complex carbohydrate content of the two variant sublines DV2 and DVl 1 and of the parental line. The DEAE-cellulose column profiles in fig. 4 show a reduced level of hyaluronic acid (HA) in the trypsin-removable material (TRM) of [3H]glucosamine-labeled DV2 cells and a greatly elevated level in the rapidly detaching DVll variant. The small amount of HA detected in DV2 is in agreement with the finding of little or no HA in the TRM of another slowly detaching variant, DV 1, independently isolated in this Laboratory [3]. The radioactivity data are presented in

table 1, which includes calculations of percentages of the parental CHO content. The rapidly detaching variant DVl 1 has 289 % of the HA of the parental cells, and the slowly detaching DV2 clone has only half the normal content. Radioactivity ascribable to HA is also elevated in the pellet of DVll following trypsin treatment and is reduced in the pellet of DV2. Although the levels of glycopeptides, heparan sulfate, and chondroitin sulfate are similar for the three lines tested, it is apparent that DVl 1 has only 66% of the parental cell level of 35Sas TRM. In a previous publication [3], the independently isolated, slowly detaching variant was found to have 305% of the parental CHO level of 35Sdetected as hepExp Cell RPS 119 (1979)

332

Barnhart,

Cox and Kraemer

aran sulfate in the residual cells (i.e., following trypsinization to release the TRM). DISCUSSION The present report confirms and extends our previous observation [3] that a variant subline of CHO cells with increased resistance to detachment synthesized little or no hyaluronic acid. Taken together with the present report concerning additional variants exhibiting either slower or more rapid detachment than the parental CHO cells, the data suggest that the HA content of the cell periphery plays a role in ease of detachment. The HA content is probably not the only determinant of ease of detachment. Data from other laboratories [2, lo] suggest that other chemical modifications of the cell periphery play a role, and Juliano’s excellent discussion of the overall situation [lo] need not be repeated here. However, our data emphasize the role of HA, at least for CHO sublines, since three variants independently selected for changes in ease of detachment all showed correlative changes in HA content. It is difficult to assess the signilicance with respect to detachability of the sulfated mucopolysaccharides; however, Culp [ 1l] has shown that heparan sulfate is an important component of the matrix materials left on the substratum when 3T3 cells are detached. Our hypothesis relating glycosaminoglycans with detachability is that

Exp Cd/ Res 119 (1979)

mediated detachment is modified by HA, which regulates access to the adhesion entities for exogenous agents such as trypsin and EGTA. The easier detachability of DVl 1 by mechanical force may be a reflection of altered HA content, sulfated constituents, or as yet unidentified determinants. It is possible that some complex carbohydrates of the cell periphery cause swelling of the “undercellular” matrix in a way that permits improved access to detachment mediators by diffusion and which modulates, perhaps non-specifically, the integrity of the cell-substratum interaction. This work was performed under the auspices of the United States Department of Energy. We wish to express our gratitude to Julie Grilly for performing the photomicrography.

REFERENCES 1. Pouyssegur, J M & Pastan, I, Proc natl acad sci US 73 (1976) 544. 2. Pouyssegur, J M, Willingham, M & Pastan, I, Proc natl acad sci US 74 (1977) 243. 3. Atherly, A G, Bamhart, B J & Kraemer, P M, J cell physiol90 (1977) 375. 4. Tjio, J & Puck, T T, J exp med 108(1958) 259. 5. Ham, R G, Exp cell res 29 (1%3) 515. 6. Puck, T T, Ciecura, S J &Robinson, A, J exp med 108 (1958) 945. 7. House, W & Waddel, A, J pathol bact 93 (1%7) 125. 8. Chanock, R M, Hayflick, L & Barile, M F, Proc nati acad sci US 48 (1%2) 41. 9. Kraemer, PM, Biochemistry 10 (1971) 1437. 10. Juliano, R L, J cell biol76 (1978) 43. 11. Gulp, L A, Rollins, B J, Buniel, J & Hitri, S (1978). Personal communication. Received June 15, 1978 Revised version received September 4, 1978 Accepted September 8, 1978