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Communication THE JOURNAL OF B~OL.OGICAL CHEMW RYVol. 265, No. 9, Issue of March 25, p. 4775-4777,199O0 1990 by The American Society for Biochemistry and MOP cular Biology , Inc.Pr in ted i n U .S .A .Alkalinization of the Lysosomes IsCorrelated with rasTransformation of Murine andHuman Fibroblasts

(Received or publication, November 1, 1990)Lian-Wei Jiang, Veronica M. MaherS,J. Just in McCorm ick+, and Melvin SchindlergFrom the Department of Biochemistry, Michigan StateUniversity, East Lansing, Michigan 48824-1316 and the$Carcinogenesi.s Laboratory, Department of Microbiologyand Public Health, Michigan State University,East Lansing, Michigan 48824

The pH of the intralysosomal compartmen t of f ibro-blasts in culture was monitored by measuring the f lu-orescence emission intensity at 530 nm of f luid phasepinocytosed f luorescein-conjugated dextrans (FITC-dextrans) excited at 488 and 457 nm. Following theprocedure of Ohkuma and Poole (Ohkuma, S., andPoole, B. (1978) Proc. Nat l. Acad. Sci. U. S. A. (1978)75, 3327-3331 ), a relationship wa s established be-tween the f luorescence emission intensity of the FITC -dextrans and pH. This correlat ion was used to deter-mine the intralysosomal apparent pH (pH,,) of a seriesof f ibroblast cultures. The mean intralysosomal pH,,values of nontransformed mouse 3T3 f ibroblasts andan infinite life-span huma n fibroblast cell strain, de s-ignated MSU -1.1, was 5.0. In dist inction that of 3T3f ibroblasts transformed to the malignant state by Kir-sten murine sarcoma virus and MSU-1.1 cells trans-formed by transfect ion of the v-Ki-ras or T24 H-rasoncogene was 6.1. These measurem ents suggest thatras transform ation results in a significan t perturba-t ion of lysosomal pH.

During recent efforts to examine the role of polypeptidegrowth factors in modulating nucleocytoplasmic transport, weexamined the accumulation of lz51-labeled epidermal growthfactor (EGF) by nontransformed murine 3T3 fibroblasts (ce llline 3T3-1) and Kirsten murine sarcoma virus (KMSV)-transformed 3T3 fibroblasts (KMSV-3T3). Administration ofleupeptin, an inhibitor of cathepsin B, caused a signif icant(-4-fold ) enhancement of lz51 -EGF accumula tion in the 3T3-1 fibroblasts, as previously reported by others (1, 2). Surpris -ingly, however, leupeptin had little influence on lz51-EGFaccumulation in KMSV-3T3 fibroblasts (-1.3-fold enhance-ment). These observations suggested that perhaps Kirsten

* This work was supported by Department of Health and HumanServices Grant GM-30158 (to M. S.) and by Department of EnergyGrant DE-FG02-87 ER 60524 (to J. J. M.). The costs of publicationof this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked aduertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J To whom all correspondence should be addressed.1 The abbreviations used are: EGF, epidermal growth factor;KMS V, Kirsten murine sarcoma virus; FITC-dextrans, fluorescein-

labele d dextrans; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesul-fonic acid.

L.-W. Jiang and M. Schindler, unpublished results.

virus transformation, a result of the K-ras oncogene, affectedsome endosomal/lysosomal activity or property that couldinfluence cathepsin B localization or its sensitivity to theinhibito r. Since previous work had demonstrated that theactivity and composition of enzymes in lysosomes could besignificantly modified by changes in intralysosomal pH (3-8),we initiated a series of experiments to examine the possibi litythat ras transformation could also modify endosomal/lysoso-ma1 activity by leading to changes in intralysosomal pH.Resu lts of our experiments in nontransformed and ras-trans-formed murine and human fibroblast lines show that rustransformation is correlated with a significant alkalinizationof the intralysosomal compartment (increasing the mean pHvalues from 5.0 to 6.1). Treatment of nontransformed 3T3cells with chloroquine raises the mean pH value to 6.3. Thisincrease in pH i s similar to that previously reported forlysosomes in mouse peritoneal macrophages following treat-ment with chloroquine (9) and in the lysosomes of humanepidermoid carcinoma A431 cells following exposure to meth-ylamine (10).EXPERIMENTAL PROCEDURES

Reagents-Fluorescein-labeled dextrans (FITC-dextrans) (M,40,000, designated 40 K) and chloroquine were obtained from Sigma .Cell Lines and Culture Conditions-The 3T3-1 fibroblast cell line,

derived from Swiss albino mice and the Kirsten murine sarcomavirus-transformed 3T3 Balb/C A31 cell line (KMSV-3T3), were ob-tained from the American Tissue Culture Collection (Rockville, MD).The infinite life-span MSU-1.1 cell strain was generated after trans-fection of foreskin-derived normal diplo id human fibroblasts with aplasmid carrying a v-myc oncogene and a selectable marker. The cellsdisplay a normal morphology and have exhibited the same neardiploi d karyotype for more than 200 popula tion doublings postcrisis.They produce small sized colonies in soft agar at a very low frequencybut do not form foci and are nontumorigenic. Transfection of MSU-1.1 cells with the pHO6Tl plasmid carrying the T24 H-ros oncogeneresulted in distinct foci of morphologica lly transformed cells (11).The progeny of cells isolated from such foci (designated MSU-l.l-T24) were shown to form rapidly growing malign ant fibrosarcomasin athymic mice (11).

In a similar fashion, transfection of MSU-1.1 cells with plasmidpK4e containing the provirus of Kirsten murine sarcoma virus in-serted into pBR322 resulted in distinct foci, and the progeny of cellsisolated from such foci (designated MSU-l.l-v-Ki-ras) were tumori-genie in athymic mice.3 3T3-1 and KMSV-3 T3 cells were plated in35-mm dia meter tissue culture plates, grown in Dulbeccos modifie dEagles mediu m supplemen ted with 10% fetal calf serum, and main-tained in a humid ified 37 C incubator with a n atmosphere of 10%COz, 90% air. The MSU-1.1, MSU-l.l-T24, and MSU-l.l-v-Ki-rascell strains were routinely cultured in Eagles m inimu m essentialmedium supplemented with 0.2 m M serine, 0.2 m M aspartate, 1.0 m Mpyruvate, 10% fetal bovine serum, 100 units/ml penicilli n, and 100fig/ml streptomycin in a humid ified 37 C incubator with an atmos-phere of 5% CO?, 95% air.

Measurement of Intralysosomal pH-Cells in exponential growthwere treated with fluoresceinated dextrans (40 K FITC-dextrans) ata concentration of 1.5 mg/m l culture mediu m when the cells weretwo to three ce ll divisions prior to confluence. The cells were incu-bated in the presence of the FITC-dextrans for 18-24 h. During thistime, the FITC-dextrans were incorporated by fluid phase pinocytosisand transferred to lysosomes. FITC-dextrans have been shown toremain stable and fluorescent in these lysosomes (9). Followingincubation, the cells were washed 3 times with their respective me-dium, lacking serum but buffered with 50 mM Hepes, pH 7.4. Meas-

3 D. G. Fry, L. D. Milam , J. E. Dillberger, V. M. Maher, and J. J.McCormick, Oncogene, submitted for publication .

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4776 Intralysosomal pH Is Affected by ras Transformationurements of f luorescence were performed in this buffered medium .The f luorescent-labeled cells were placed on the stage of an ACAS470 interact ive laser cytome ter (Meridian Instrume nts, Inc., Okem os,MI) to image the intracellular distr ibut ion and intensity of lysosomalf luorescence. As previously described (13, 14), the automated stagemove s in 2-Km steps in a two-dimensional raster pattern, past amicroscope object ive (X 40) that focuses the argon ion laser (5 watts)excitat ion beam (f irst at 457 nm (200 mil l iwatts) and then at 488 nm(200 mil l iwatts)) to a l-Nm diameter beam on the sample. A photo-mult ipl ier tube captures the emission intensit ies at 530 nm for bothexcitat ion wavelengths at each addressed excitat ion point in thesample. The emitted intensit ies for each excitat ion wavelength arethen color-coded and displayed on a cathode ray tube as false colorimages of lysosomal localized f luorescence. Th e f luorescence emissionintensity/pixel at 530 nm for each excitat ion wavelength was obtainedby calculat ing the total f luorescence in a cell and dividing by thenumber of pixels that form the cell image. As described in greaterdetai l under Results and Discussion, the f luorescence emissionintensity after excitat ion at 488 nm w as divided by the intensity/pixel af ter excitat ion at 457 nm to give an intensity rat io. T o minimizephotobleaching, measurem ents were f irst made at 457 nm and thenat 488 nm. Color has been converted to shades of gray in Fig. 1.

Generating a Standard Curve Relatin g Fluorescence to pH--100 ~1of a 40 K FITC-dextran solut ion (1 mg/m l dextrans in water) w asadded to 1 ml of buffer at the desired pH. An 80-~1 drop of thisf luorescent solut ion was placed in a bounded compa rtment on a

e

FIG. 1. Intralysosomal PH.,, as measured by the fluores-cence of fluid phase pinocytosed FITC-dextrans. Treatment ofcultured 3T3-1 and KMSV -3T3 cells with 40 K FITC-de xtrans wasperformed as described under Experimental Procedures and re-sulted in the lysosomal incorporation of the labeled dextrans. Flu-orescent images of f ibroblasts at Xemission 530 nm and Xe xEitelion 488nm are observed for 3T3-1 (a), KMSV -3T3 (c), and 3T3-1 incubatedin 30 pM chloroquine (e), while the same cells viewed a t h,, i , , i , , = 530nm and XPXeital iUn 457 nm are observed in b, d, an d f for 3T3-1,KMSV -3T3, and 3T3-1 incubated in 30 pM chloroquine, respect ively.Gray spectrum to the right of the images represents the relat ionbetween shades of gray and f luorescence intensity. Arrow in imagesis a cursor ut i l ized to mark areas of f luorescence intensity measure-ments .

1L-----J 6 7PH

F I G. 2. Fluorescence standard curve for obtain ing intraly-sosomal PH.,,. The f luorescence rat io of FITC-dex tran emission attwo dif ferent excitat ion wavelengths is plotted as a funct ion of pHfor six samples in buffers of known pH. Th is cu rve may then beut i l ized in conjunct ion with f luorescence intensity rat ios from FITC-dextran measu rements in cells to obtain intralysosomal pH,, , .

T A B L E IIntralysosomal pH,, in nontransformed and ras-transformed

fibroblastsCel l l i ne R Calculated PH., ,

MSU -1.1 0.89 f 0.10 (41) 5. 03T3-1 0.85 + 0.08 (5) 5. 03T3-1 + 30 PM chloroquine 1.68 + 0.11 (3) 6. 4KMSV-3T3 1.32 + 0.12 (4) 5.8MSU-l. l-v-Ki-ras 1.65 f 0.22 (25) 6.3MSU-l. l-T24 1.67 & 0.15 (13) 6.3a R = &a - ~~~ea)/(~~~, - IB1 5 1 ) .*Mean + SD. Numbe r of experimen ts.

microscope sl ide. Drop containment was maintained by placing thedrop in a circle made with melted w ax. The laser was then focused tothe sl ide/ l iquid interface, and an image was generated as describedfor the dextran-loaded cells. To permit the use of the same photo-mult ipl ier and excitat ion intensity sett ings between pH standards,appropriate di lut ions were made with the buffered samples to main-tain equivalent f luorescent intensit ies between samples.RESULTS AND DISCUSSION

As a result o f work by Marl in and Lindquist (15) showingthat the f luorescence emission of f luorescein could be affectedby pH, O hkuma and Poole (9) developed a method to deter-mine the intravesicular PH., of lysosom es in viable cells inculture. In their procedure, f luoresceinated dextrans (40 KFITC-dex trans) serve as lysosome-sp ecif ic probes of intraly-sosoma l pH,, (see Experimental Procedures). 3T3-1 andKMSV -3T3 cells labeled with these FITC-de xtrans and digi-tal ly imaged at excitat ion wavelengths 457 and 488 nm areshown in Fig. 1. Punctate gray points represent lyso some swith incorporated FITC-de xtrans. As noted by Ohkuma andPoole (9), the alkaline excitat ion spectrum of FITC-de xtransis dominated by a peak at 495 nm which disappears as the pHis lowered, yielding two new peaks of fluorescence excitationat wavelengths of 480 and 450 nm. Monitoring a single emis-sion at these two excitation wavelengths may, therefore, beemployed in conjunction with results obtained with a seriesof buffers (standard curve) to calculate values of intralysoso-ma1 PH. , , in living cells (9, 12). Since the exact amount ofirradiated FITC-dextran in a cel l is not known, we have useda procedure that measures the fluorescence emission intens ity

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Intralysosomal pH Is Affected by ras Transformation 4777at 530 nm follow ing excita tion at 488 and 457 nm and takesthe ratio between these two values. Such ratio measurementsare FITC-dextran concentration-independent. In the presentstudy, the data are represented as an intens ity ratio, R = (I488- IB,#h57 - IBds,), where Z4=and L7 represent the fluores-cence emission intensity/pixel at 530 nm automatically cal-culated from cel l images generated at excitation wavelengthsof 457 and 488 nm (Ar laser), respectively. ZBa and Z& arebackgrounds of cellular autofluorescence at each excitationwavelength, measured over small regions of the ce ll containingno lysosomes. To determine the relationship between theseratios and the PH.,, of the lysosoma l compartment of thecells, a standard curve was generated (Fig. 2).The intensity ratio, R, of FITC-dextrans dissolved in aseries of buffers yielded a linear relationship between fluores-cence emission intensity and pH over a pH range from 5 to7. We employed this relationship to determine the intravesic-ular PH.,, of cellular lysosomes from the fluorescence emis-sion of lysosomal images obtained with the cell lines shownin Fig. 1. The data for these mouse cell lines are listed inTable I along with the results of simila r measurements per-formed on nontransformed human fibroblasts (MSU-1.1) andhuman fibroblasts transformed with v-Ki- ras (M SU-l.l-v -Ki-roe) and the T2 4 H-rczs oncogene (MSU-l. l-T24). Asshown in Table I, all ras-transformed cell lines demonstratedan abnormally high intralysosomal pH,,,. The difference inpHepp between the KMSV-3T3 cells and MSU-l.l-v-K i-rascells may simply reflect the fact that MSU-l.l-v-Ki -ras cellsare clonally derived and all of them exhibit the identical levelof Ki-ras expression.3 KMSV-3T3 is a heterogenous popula-tion of transformed cells which may exhibit varying levels ofv-Ki- ras expression. The degree of alkalinization observedwith these rus-transformed ce lls is comparable to the degreeof alkalinization observed in 3T3-1 cell s treated with chloro-quine (Table I) and by others following treatment of mousecells with chloroquine (9) or methylamine (10).

The results of the present study suggest a means by whichra.s transformation could affect some aspect of endosomal/lysosomal organization and function. Since the maintenanceof an acidic intralysosomal pH is essential for correct receptortrafficking, receptor sorting, endocytosis, and lysosomal bio-synthesis (7), alkalinization of the intralysosomal compart-ment and perhaps endosomal compartments, as a resul t ofras oncogene transformation, could have major consequencesfor all cellular signaling and proliferative pathways.

Acknowledgment-We express our thanks to Asmina Jiwa of Me-ridian Instruments (Okemo s, MI) for assistance with prel iminarymeasurements .

1.2.3.4.5.6.7.8.9.

10 .11 .12 .13 .14 .15 .

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