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Alkalinization of the Lysosomes Is Correlated with ras Transformation of Murine and Human Fibroblasts

THE JOURNAL OF B~OL.OGICAL CHEMWRY Vol. 265, No. 9, Issue of March 25, p. 4775-4777,199O ecular Biology, Inc. 0 1990 by The American Society for Biochemistry and MO P Printed in U.S.A.

virus transformation, a result of the K-ras oncogene, affected some endosomal/lysosomal activity or property that could influence cathepsin B localization or its sensitivity to the inhibitor. Since previous work had demonstrated that the activity and composition of enzymes in lysosomes could be significantly modified by changes in intralysosomal pH (3-8), (Receivedfor publication, November 1, 1990) we initiated a series of experiments to examine the possibility that ras transformation could also modify endosomal/lysosoLian-Wei Jiang, Veronica M. MaherS, ma1 activity by leading to changes in intralysosomal pH. J. Justin McCormick+, and Melvin Schindlerg Results of our experiments in nontransformed and ras-transFrom the Department of Biochemistry, Michigan State formed murine and human fibroblast lines show that rus University, East Lansing, Michigan 48824-1316 and the transformation is correlated with a significant alkalinization $Carcinogenesi.s Laboratory, Department of Microbiology and Public Health, Michigan State University, of the intralysosomal compartment (increasing the mean pH East Lansing, Michigan 48824 values from 5.0 to 6.1). Treatment of nontransformed 3T3 cells with chloroquine raises the mean pH value to 6.3. This The pH of the intralysosomal compartment of fibroincrease in pH is similar to that previously reported for blasts in culture was monitored by measuring the flulysosomes in mouse peritoneal macrophages following treatorescence emission intensity at 530 nm of fluid phase ment with chloroquine (9) and in the lysosomes of human pinocytosed fluorescein-conjugated dextrans (FITCepidermoid carcinoma A431 cells following exposure to methdextrans) excited at 488 and 457 nm. Following the ylamine (10).
procedure of Ohkuma and Poole (Ohkuma, S., and Poole, B. (1978) Proc. Natl. Acad. Sci. U. S. A. (1978) 75, 3327-3331), a relationship was established between the fluorescence emission intensity of the FITCdextrans and pH. This correlation was used to determine the intralysosomal apparent pH (pH,,) of a series of fibroblast cultures. The mean intralysosomal pH,, values of nontransformed mouse 3T3 fibroblasts and an infinite life-span human fibroblast cell strain, designated MSU-1.1, was 5.0. In distinction that of 3T3 fibroblasts transformed to the malignant state by Kirsten murine sarcoma virus and MSU-1.1 cells transformed by transfection of the v-Ki-ras or T24 H-ras oncogene was 6.1. These measurements suggest that ras transformation results in a significant perturbation of lysosomal pH.

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During recent efforts to examine the role of polypeptide growth factors in modulating nucleocytoplasmic transport, we examined the accumulation of lz51-labeled epidermal growth factor (EGF) by nontransformed murine 3T3 fibroblasts (cell line 3T3-1) and Kirsten murine sarcoma virus (KMSV)transformed 3T3 fibroblasts (KMSV-3T3). Administration of leupeptin, an inhibitor of cathepsin B, caused a significant (-4-fold) enhancement of lz51-EGF accumulation in the 3T31 fibroblasts, as previously reported by others (1, 2). Surprisingly, however, leupeptin had little influence on lz51-EGF accumulation in KMSV-3T3 fibroblasts (-1.3-fold enhancement). These observations suggested that perhaps Kirsten
* This work was supported by Department of Health and Human Services Grant GM-30158 (to M. S.) and by Department of Energy Grant DE-FG02-87 ER 60524 (to J. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance 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; KMSV, Kirsten murine sarcoma virus; FITC-dextrans, fluoresceinlabeled dextrans; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. L.-W. Jiang and M. Schindler, unpublished results.

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 sarcoma virus-transformed 3T3 Balb/C A31 cell line (KMSV-3T3), were obtained from the American Tissue Culture Collection (Rockville, MD). The infinite life-span MSU-1.1 cell strain was generated after transfection of foreskin-derived normal diploid human fibroblasts with a plasmid carrying a v-myc oncogene and a selectable marker. The cells display a normal morphology and have exhibited the same near diploid karyotype for more than 200 population doublings postcrisis. They produce small sized colonies in soft agar at a very low frequency but do not form foci and are nontumorigenic. Transfection of MSU1.1 cells with the pHO6Tl plasmid carrying the T24 H-ros oncogene resulted in distinct foci of morphologically transformed cells (11). The progeny of cells isolated from such foci (designated MSU-l.lT24) were shown to form rapidly growing malignant fibrosarcomas in athymic mice (11). In a similar fashion, transfection of MSU-1.1 cells with plasmid pK4e containing the provirus of Kirsten murine sarcoma virus inserted into pBR322 resulted in distinct foci, and the progeny of cells isolated from such foci (designated MSU-l.l-v-Ki-ras) were tumorigenie in athymic mice.3 3T3-1 and KMSV-3T3 cells were plated in 35-mm diameter tissue culture plates, grown in Dulbeccos modified Eagles medium supplemented with 10% fetal calf serum, and maintained in a humidified 37 C incubator with an atmosphere of 10% COz, 90% air. The MSU-1.1, MSU-l.l-T24, and MSU-l.l-v-Ki-ras cell strains were routinely cultured in Eagles minimum essential medium supplemented with 0.2 mM serine, 0.2 mM aspartate, 1.0 mM pyruvate, 10% fetal bovine serum, 100 units/ml penicillin, and 100 fig/ml streptomycin in a humidified 37 C incubator with an atmosphere of 5% CO?, 95% air. Measurement of Intralysosomal pH-Cells in exponential growth were treated with fluoresceinated dextrans (40 K FITC-dextrans) at a concentration of 1.5 mg/ml culture medium when the cells were two to three cell divisions prior to confluence. The cells were incubated in the presence of the FITC-dextrans for 18-24 h. During this time, the FITC-dextrans were incorporated by fluid phase pinocytosis and transferred to lysosomes. FITC-dextrans have been shown to remain stable and fluorescent in these lysosomes (9). Following incubation, the cells were washed 3 times with their respective medium, lacking serum but buffered with 50 mM Hepes, pH 7.4. Meas3 D. G. Fry, L. D. Milam, J. E. Dillberger, V. M. Maher, McCormick, Oncogene, submitted for publication. and J. J.



pH Is Affected by ras Transformation

urements of fluorescence were performed in this buffered medium. The fluorescent-labeled cells were placed on the stage of an ACAS 470 interactive laser cytometer (Meridian Instruments, Inc., Okemos, MI) to image the intracellular distribution and intensity of lysosomal fluorescence. As previously described (13, 14), the automated stage moves in 2-Km steps in a two-dimensional raster pattern, past a microscope objective (X 40) that focuses the argon ion laser (5 watts) excitation beam (first at 457 nm (200 milliwatts) and then at 488 nm (200 milliwatts)) to a l-Nm diameter beam on the sample. A photomultiplier tube captures the emission intensities at 530 nm for both excitation wavelengths at each addressed excitation point in the sample. The emitted intensities for each excitation wavelength are then color-coded and displayed on a cathode ray tube as false color images of lysosomal localized fluorescence. The fluorescence emission intensity/pixel at 530 nm for each excitation wavelength was obtained by calculating the total fluorescence in a cell and dividing by the number of pixels that form the cell image. As described in greater detail under Results and Discussion, the fluorescence emission intensity after excitation at 488 nm was divided by the intensity/ pixel after excitation at 457 nm to give an intensity ratio. To minimize photobleaching, measurements were first made at 457 nm and then at 488 nm. Color has been converted to shades of gray in Fig. 1. Generating a Standard Curve Relating Fluorescence to pH--100 ~1 of a 40 K FITC-dextran solution (1 mg/ml dextrans in water) was added to 1 ml of buffer at the desired pH. An 80-~1 drop of this fluorescent solution was placed in a bounded compartment on a


6 PH 7

FIG. 2. Fluorescence standard curve for obtaining intralyThe fluorescence ratio of FITC-dextran emission at sosomal PH.,,. two different excitation wavelengths is plotted as a function of pH for six samples in buffers of known pH. This curve may then be utilized in conjunction with fluorescence intensity ratios from FITCdextran measurements in cells to obtain intralysosomal pH,,,.
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and ras-transformed
R Calculated PH.,,

Cell line


in nontransformed fibroblasts

MSU-1.1 3T3-1 3T3-1 + 30 PM chloroquine KMSV-3T3 MSU-l.l-v-Ki-ras MSU-l.l-T24

a R = &a - ~~~ea)/(~~~, IB151).

0.89 0.85 1.68 1.32 1.65 1.67

f + + + f &

0.10 (41) 0.08 (5) 0.11 (3) 0.12 (4) 0.22 (25) 0.15 (13)

5.0 5.0 6.4 5.8 6.3 6.3

*Mean + SD. Number of experiments. microscope slide. Drop containment was maintained by placing the drop in a circle made with melted wax. The laser was then focused to the slide/liquid interface, and an image was generated as described for the dextran-loaded cells. To permit the use of the same photomultiplier and excitation intensity settings between pH standards, appropriate dilutions were made with the buffered samples to maintain equivalent fluorescent intensities between samples.

FIG. 1. Intralysosomal
cence of fluid phase

Treatment of cultured 3T3-1 and KMSV-3T3 cells with 40 K FITC-dextrans was performed as described under Experimental Procedures and resulted in the lysosomal incorporation of the labeled dextrans. Fluorescent images of fibroblasts at Xemission 530 nm and XexEitelion 488 = = nm are observed for 3T3-1 (a), KMSV-3T3 (c), and 3T3-1 incubated in 30 pM chloroquine (e), while the same cells viewed at h,,i,,i,, = 530 nm and XPXeitaliUn 457 nm are observed in b, d, and f for 3T3-1, = KMSV-3T3, and 3T3-1 incubated in 30 pM chloroquine, respectively. Gray spectrum to the right of the images represents the relation between shades of gray and fluorescence intensity. Arrow in images is a cursor utilized to mark areas of fluorescence intensity measurements.

PH.,, pinocytosed

as measured FITC-dextrans.




As a result of work by Marlin and Lindquist (15) showing that the fluorescence emission of fluorescein could be affected by pH, Ohkuma and Poole (9) developed a method to determine the intravesicular PH., of lysosomes in viable cells in culture. In their procedure, fluoresceinated dextrans (40 K FITC-dextrans) serve as lysosome-specific probes of intralysosomal pH,, (see Experimental Procedures). 3T3-1 and KMSV-3T3 cells labeled with these FITC-dextrans and digitally imaged at excitation wavelengths 457 and 488 nm are shown in Fig. 1. Punctate gray points represent lysosomes with incorporated FITC-dextrans. As noted by Ohkuma and Poole (9), the alkaline excitation spectrum of FITC-dextrans is dominated by a peak at 495 nm which disappears as the pH is lowered, yielding two new peaks of fluorescence excitation at wavelengths of 480 and 450 nm. Monitoring a single emission at these two excitation wavelengths may, therefore, be employed in conjunction with results obtained with a series of buffers (standard curve) to calculate values of intralysosoma1 PH.,, in living cells (9, 12). Since the exact amount of irradiated FITC-dextran in a cell is not known, we have used a procedure that measures the fluorescence emission intensity


pH Is Affected by ras Transformation


at 530 nm following excitation at 488 and 457 nm and takes the ratio between these two values. Such ratio measurements are FITC-dextran concentration-independent. In the present study, the data are represented as an intensity ratio, R = (I488 - IBds,), where Z4=and L7 represent the fluores- IB,#h57 cence emission intensity/pixel at 530 nm automatically calculated from cell images generated at excitation wavelengths of 457 and 488 nm (Ar laser), respectively. ZBa and Z& are backgrounds of cellular autofluorescence at each excitation wavelength, measured over small regions of the cell containing no lysosomes. To determine the relationship between these ratios and the PH.,, of the lysosomal compartment of the cells, a standard curve was generated (Fig. 2). The intensity ratio, R, of FITC-dextrans dissolved in a series of buffers yielded a linear relationship between fluorescence emission intensity and pH over a pH range from 5 to 7. We employed this relationship to determine the intravesicular PH.,, of cellular lysosomes from the fluorescence emission of lysosomal images obtained with the cell lines shown in Fig. 1. The data for these mouse cell lines are listed in Table I along with the results of similar measurements performed on nontransformed human fibroblasts (MSU-1.1) and human fibroblasts transformed with v-Ki-ras (MSU-l.l-vKi-roe) and the T24 H-rczs oncogene (MSU-l.l-T24). As shown in Table I, all ras-transformed cell lines demonstrated an abnormally high intralysosomal pH,,,. The difference in pHepp between the KMSV-3T3 cells and MSU-l.l-v-Ki-ras cells may simply reflect the fact that MSU-l.l-v-Ki-ras cells are clonally derived and all of them exhibit the identical level of Ki-ras expression.3 KMSV-3T3 is a heterogenous population of transformed cells which may exhibit varying levels of v-Ki-ras expression. The degree of alkalinization observed with these rus-transformed cells is comparable to the degree of alkalinization observed in 3T3-1 cells treated with chloroquine (Table I) and by others following treatment of mouse cells with chloroquine (9) or methylamine (10).

The results of the present study suggest a means by which ra.s transformation could affect some aspect of endosomal/ lysosomal organization and function. Since the maintenance of an acidic intralysosomal pH is essential for correct receptor trafficking, receptor sorting, endocytosis, and lysosomal biosynthesis (7), alkalinization of the intralysosomal compartment and perhaps endosomal compartments, as a result of ras oncogene transformation, could have major consequences for all cellular signaling and proliferative pathways.

ridian Instruments measurements. 1. 2. 3. 4.

express our thanks to Asmina Jiwa of Me(Okemos, MI) for assistance with preliminary

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