Pyridoxine Resistance in a Rat Hepatoma Cell Line1

[CANCER RESEARCH 42, 2362-2370. June 1982] 0008-54 72/82 /0042-OOOOS02.00 Pyridoxine Resistance in a Rat Hepatoma Cell Line1

Dennis M. DiSorbo, Laurie G. Paavola, and Gerald Litwack '

Fels Research Institute [D. M. D.. G. L.] and Departments of Biochemistry Â¡D.M. D., G. L.J and Anatomy [L. G. P.I, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

ABSTRACT vitamin B6 but still transported the vitamin into the cell and metabolized it to the active vitamer, PLP.4 The natural vitamin, pyridoxine, in the millimolar range is In the present communication, we report the establishment toxic to cultured rat hepatoma cells. A pyridoxine-resistant of a pyridoxine-resistant hepatoma cell line. The newly estab Fu5-5 rat hepatoma cell line was established by a stepwise lished cell line is shown to be resistant to killing by 5 mM increase in the concentration of pyridoxine in the medium. The pyridoxine-supplemented medium. Resistance appears to be newly established cell line, referred to as clone 10 (CI. 10), is the result of a mutation other than one in permeability of the resistant to killing by pyridoxine in concentrations up to 5 rrtM. Saturation kinetics for the uptake of [3H]pyridoxine into Fu5-5 membrane to pyridoxine or one in the pyridoxal kinase enzyme system. The mutation may involve some feature of mitochon and CI. 10 cells revealed that Fu5-5 cells take up 10 times more drial development. The possibility of using the pyridoxine-re [3H]pyridoxine than do CI. 10 cells. Whereas the Vmaxvalue for sistant cell line to'investigate the regulation of several key the uptake of [3H]pyridoxine was the same for both cell lines, metabolite enzymes by PLP is discussed. the apparent Km for the CM 0 cells was 12.5 juM compared to 0.71 /Â¿Mfor the Fu5-5 cells. However, intracellular levels of pyridoxal 5'-phosphate were 69% higher in CI.10 cells than in MATERIALS AND METHODS the parental line. The resistant line is neither a permeability Cell Culture Conditions mutant nor deficient in pyridoxal kinase. CI. 10 cells contain 37% more adenosine 5'-triphosphate than do Fu5-5 cells and Eagle's MEM (Grand Island Biological Co., Grand Island, N. Y.) have a mitochondrial volume that is 50% greater than that of supplemented with 10% calf serum (M. A. Bioproducts, Walkersville, the parental line. In the absence of pyridoxine in the medium, Md.), gentamicin (50 jig/ml; M. A. Bioproducts), and 2 mM glutamine were used for maintaining Fu5-5 rat hepatoma cells. Pyridoxine-resist CI. 10 cells revert to parental type with respect to pyridoxine ant Fu5-5 rat hepatoma cells were maintained in the same medium but uptake but not with respect to resistance to killing. They also supplemented with a final concentration of 5 mM neutralized pyridoxine maintain an enlarged mitochondrial volume. Thus, increased HCI (Sigma Chemical Co., St. Louis, Mo.). Cells were grown in Falcon mitochondrial volume may be related to the development of T-75 sq cm flasks and maintained at 37Â°in a humidified atmosphere resistance to high levels of pyridoxine. of 5% CO2 in air. Establishment of Pyridoxine-resistant Cell Line INTRODUCTION Fu5-5 rat hepatoma cell line was derived from the H4-II-E-C3 line We have demonstrated that high doses of vitamin B6 when (4). Cells were plated in T-75 sq cm flasks and cultured for 2 weeks in added to tissue culture medium prevented the proliferation of growth medium supplemented with 0.5 mM neutralized pyridoxine. The Fu5-5 rat hepatoma cells (5). Although the mechanism by pyridoxine concentration was then increased in increments of 0.5 mM which vitamin B6 inhibited cell growth was not ascertained, every 2 weeks until a final concentration of 5 mM was reached. experimentation did reveal that cells cultured for 6 hr in vitamin Since the increased concentrations of pyridoxine caused cell death Be-enriched medium took up and incorporated less [3H]thymi- (trypan blue exclusion test), the remaining viable cells, resistant to 5 dine than did control cultures. Further studies indicated that mM pyridoxine, formed distinct colonies. These colonies were subse quently cloned in T-25 sq cm flasks and maintained in culture medium the intracellular levels of ATP were elevated in cultures exposed supplemented with 5 mM pyridoxine. Of the colonies cloned, one to the vitamin for short periods of time. If cells were cultured in survived and was established in culture. This pyridoxine-resistant cell high-vitamin B6-supplemented medium for more than 4 days, line is referred to as Fu5-5 PR clone 10 and is abbreviated as CI. 10 cell death occurred. elsewhere in this paper. Since Fu5-5 rat hepatoma cells were unable to sustain Cellular Uptake of [3H]Pyridoxine growth in culture medium supplemented with high doses of vitamin B6, studies to elucidate how the vitamin regulates cell Pyridoxine resistant CI. 10 and control Fu5-5 cells were cultured in growth were unsuccessful. Therefore, it seemed necessary to serum-free MEM without pyridoxine (No. 78-0707; Grand Island Bio develop a cell line that was resistant to killing by high doses of logical Co.) for 48 hr before use. At the start of the experiment, medium was aspirated off, and 10 ml of pyridoxine-free, serum-free MEM were ' Supported by Research Grants PCM-78-25767 from the National Science added to each T-75 sq cm flask. To this medium was added [3H]- Foundation; AM-13531 from the National Institute of Arthritis. Metabolism, and pyridoxine (specific activity, 1.5 Ci/mmol; Amersham-Searle Corp.) to Digestive Diseases. NIH; 584-038 88L. an Institutional Grant from the American Cancer Society; and CA 12227 from the National Cancer Institute. NIH, to the a final concentration of 1.33 ftM. Flasks were incubated for various Fels Research Institute. times. At the end of each time period, the medium was removed, and 2 Postdoctoral Trainee, Grant T32-AM-07162 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, NIH, to the Department of Bio 4 The abbreviations used are: PLP, pyridoxal 5'-phosphate; MEM, minimum essential medium; PL, pyridoxal; PBS, Dulbecco's phosphate-buffered saline chemistry. Present address: Nassau Hospital Oncology Center, 222 Front Street, Suite 300, MineÃ³la, N. Y. 11501. (CaCI2, 0.88 mM; MgCI2, 0.5 mM; KCI, 2.68 mM; KH2POt, 1.5 mM; NaCI, 136.7 ' To whom requests for reprints should be addressed. mM; NaÃ®HPCu,8.8 mM); TEM, transmission electron microscopy; SEM, scanning Received December 23. 1981 ; accepted March 10, 1982. electron microscopy.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1982 American Association for Cancer Research. Pyridoxine Res/stance in Hepatoma Cells the cells were washed 3 times in PBS, and then dissolved in 3.0 ml of up on coated copper grids. These sections were stained with lead 1 N NaOH. For the determination of [3H]pyridoxine uptake as a function citrate for 2 min (16) or with uranyl acetate for 5 to 15 min followed by of its concentration, the protocol was the same as above. Radioactivity lead citrate and examined in a Phillips 300 electron microscope. was measured in a liquid scintillation counter as described previously SEM. To detect if the surface of Fu5-5 and CI.10 cells differed in (20). their topography, 5 samples of each cell line were examined after preparation for SEM. As for TEM, the cells were plated in Aclar dishes, Determination of PL and PLP allowed to grow for 24 hr, and then fixed as described above. After fixation, the cultures, still in Aclar dishes, were dried using absolute The determination of the intracellular levels of both PL and PLP was ethanol and liquid CO2 in a Sorvall critical-point drying apparatus carried out essentially by the procedure of Wada and Snell (19) with (DuPont Instruments-Sorvall Biomedicai Div. DuPont Co., Wilmington, the following modifications. Cells grown for 48 hr in serum-free medium Del.) and attached to aluminum stubs with double-sided tape. They were trypsinized off the substratum (0.1 % trypsin-0.02% EDTA in PBS were then coated with a 15- to 20-nm layer of gold in a DentÃ³n Vacuum without Ca2+ or Mg2+) and placed into 15-ml glass conical tubes. The Desk-1 sputter coaler (DentÃ³n Vacuum, Inc., Cherry Hill, N. J.) and cells were washed 3 times in PBS, and the final supernatant fraction examined in an ETEC Autoscan scanning electron microscope at an was discarded. Cold 10% (w/v) trichloroacetic acid was added to the accelerating voltage of 20 kV. Since the sample was disc shaped, 5 cell pellet to a final volume of 0.95 ml. The mixture was gently vortexed, photographs were taken of each of its quadrants for each specimen. kept on ice for 30 min, and centrifuged for 10 min in a Dynac tabletop Polaroid PN-55 film was used for photographing the samples. centrifuge at maximum speed. The supernatant was removed, 0.05 ml Stereology. Simple inspection of the TEMs suggested that CI. 10 of phenylhydrazine reagent was added to it (phenylhydrazine hydro- cells contained an increased mitochondrial compartment as compared chloride, 2 g dissolved in 100 ml of 36 N H2SO4) and the mixture was to the parental cell line. Stereology was used to examine this possibility. kept at 4Â°for 30 min. This procedure allows for the determination of The CM OR line was analyzed in a similar way. PLP in the presence of PL. For the measurement of PL, the reaction All of the samples examined were processed simultaneously for mixture was heated to 60Â° for 20 min, cooled at room temperature, TEM. Four blocks of each category, Fu5-5, CI.10, and CI.1 OR, were and assayed 10 min later. This procedure measures both PL and PLP chosen at random. To avoid counting the same cell more than once, concentrations. By subtracting the PLP measured at 4Â° from that only one thin section was selected from each block and examined. measured in the heated mixture (PL plus PLP), a value for the PL Each section was scanned using a nonrandom pattern, and the first 16 concentration can be obtained. All readings were made at 410 nm. cells that met 2 previously established criteria (14, 15) were photo graphed. Each negative was enlarged photographically to 15,700 ATP Levels in Fu5-5, CI.10, and CI.10R Cells diameters. The fraction of the cell occupied by mitochondria was determined by a point-counting method (21) in which a transparent Fu5-5, CI.10, and CI.10R cells were trypsinized off the substratum overlay bearing a grid pattern that consisted of 108 test points was and placed into 15-ml conical tubes containing MEM. The cell suspen superimposed over the photographic print. The points lying over mito sion was centrifuged, and the supernatant fraction was discarded. To chondria and those over the remaining cytoplasm and nuclei were the pellet was added 1.0 ml of serum-free MEM, and a cell suspension tabulated. For each cell line, 64 cells were photographed and analyzed. was made by gently vortexing the mixture. Cell suspension (0.2 ml) Since nuclei were included in the fields, the data are expressed as was mixed with an equal volume of nucleotide releasing reagent (Lumac percentage of cell volume (fractional cell volume). Data were analyzed Systems, Inc., Titusville, Fla.), and the released ATP was then mea with a one-way analysis of variance. The Newman-Keul test was used sured in a Luminometer M 1070 (Lumac Systems, Inc.) in the presence to locate significant differences among groups. of the bioluminescence reagent LUMIT. LUMIT is a highly purified luciferin-luciferase reagent for sensitive measurement of ATP. ATP concentration is expressed as nmol/mg protein. RESULTS Effect of Pyridoxine on the Growth of Fu5-5 Rat Hepatoma Protein Analysis Cells in Culture. Little is known of the growth characteristics Protein concentration was estimated by the method of Lowry ef al. of Fu5-5 rat hepatoma cells cultured in various vitamin B6 (11). concentrations. Therefore, a growth profile was constructed in which cell density was measured as a function of varying Morphology and Stereology concentrations of pyridoxine in the medium. Results of this TEN!. Fu5-5 and CI. 10 cells were prepared for TEM to determine if experiment are shown in Chart 1. Cells that had been cultured alterations in the mitochondrial population were correlated with the previously for 24 hr in MEM were switched to MEM containing increased levels of ATP seen in CI.10 cells. various concentrations of pyridoxine. Cells cultured in the Cells were plated onto Aclar dishes (12), previously coated with rat absence of pyridoxine showed no cell growth and in fact were tail tendon collagen (1 ), and cultured as described above. Prior to dying. Cell growth was optimal at a pyridoxine concentration of fixation, the cultures were washed 3 times with warm serum-free MEM 5 UM. Above this pyridoxine concentration, cell growth slowed; and then fixed for 15 min at 37Â° with 2% glutaraldehyde-0.1 M at a pyridoxine concentration of 5 to 10 rriM, cell death oc cacodylate containing 5% sucrose at pH 7.4. Fixation was continued curred. at 4Â°for 1.75 hr in fresh fixative. After fixation, the cultures were rinsed Establishment of a Pyridoxine-resistant Cell Line. Chart 1 in 4 changes of cold buffer (0.1 M cacodylate containing 5% sucrose), indicates that high doses of pyridoxine severely retard cell postfixed in 1.33% OsCvO.067 M cacodylate with 5% sucrose for 1 growth and, at concentrations of 5 mivi and above, cause cell hr, rinsed in buffer, stained en bloc with cold buffered uranyl acetate (6) for 1 hr at pH 6 in the dark, dehydrated in a series of graded death. Therefore, in an attempt to establish a cell line resistant ethanols, embedded in epoxy resin (17), and polymerized for 24 hr at to the killing effect of pyridoxine, a protocol was implemented 70Â°. Areas (1 cu mm) of the embedded cultures were cut out using a whereby cells were exposed to increasing concentrations of jeweler's saw and were oriented parallel to the long axis of the cells by pyridoxine over a prolonged period of time (see "Materials and Methods"). A stepwise increase in the concentration of pyri attaching them to a block previously polymerized with epoxy glue. Pale gold to silver thin sections were cut with a diamond knife and picked doxine in the medium was used. After exposure to the highest

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1982 American Association for Cancer Research. D. M. DiSorbo et al. trated only one-tenth as much [3H]pyridoxine after 60 min of incubation as did Fu5-5 cells (Chart 2). The resistant CI. 10 cells undoubtedly represent a selection of a preexisting mutant U 10 produced by the selective pressure of the high pyridoxine n dose. At first, these cells appeared to be permeability mutants. A reversion of the permeability phenotype was tested by re o moving CI.10 cells from the high pyridoxine-supplemented 0 medium and placing them in a pyridoxine-free medium. Cells were then analyzed daily for their ability to take up the B6 vitamer. As shown in Chart 3, CI.10 cells increased their daily uptake of [3H]pyridoxine and on Day 4 reached a level of J pyridoxine uptake that was approximately 85% of controls. IL Therefore, the mechanism of resistance of CI. 10 cells to high J levels of pyridoxine was not due to a permeability mutation of J 111 the parental cell line. However, cells that had reverted with U respect to pyridoxine permeability still remained resistant to B killing. U â€¢ Growth Rate of Fu5-5 and CI.10R Cells in 5 mM Pyridoxine Medium. CI. 10 cells that had been placed in pyridoxine-free Z MEM were subsequently plated in MEM, cultured for an addi O.OOB O.B tional 2 months, and then analyzed for their ability to concen O.OOSS trate [3H]pyridoxine. These cells took up approximately the same amount of [3H]pyridoxine as the Fu5-5 cells (data not mM PYRIDOXINE shown) and were classified as CI.10 revertants (CI.1 OR). Chart 1. Effect of various pyridoxine concentrations on the growth of Fu5-5 If the CI.1 OR had reverted back to the parental cell type rat hepatoma cells in culture. Fu5-5 rat hepatoma cells were plated in T-75 sq cm flasks (1 x 10e cells/flask) and cultured for 24 hr in MEM. After this (Fu5-5), they should again be sensitive to the killing effect of incubation period, cells were changed into MEM containing various concentra high, doses of pyridoxine in the medium. To test this possibility, tions of pyridoxine and cultured for an additional 72 hr. Cell density was measured Fu5-5 and CI.1 OR cells (1 x 106 cells/flask) were cultured in by counting cells in a hemocytometer in the presence of trypan blue. Results are the means of 3 T-75 sq cm flasks/point. MEM with or without 5 mM pyridoxine. Cultures were grown for 72 hr and then harvested and counted. The Fu5-5 cells grown in the absence of 5 HIM pyridoxine reached 64 x 106 cells/ flask (Table 1). Fu5-5 cells grown in the presence of 5 mM pyridoxine showed essentially no growth; instead, they dis LU played signs of cell death. In contrast to the Fu5-5 cells, CI.1 OR cells proliferated at the same rate in either medium. However, it is interesting to note that the overall growth rate of the CI.1 OR cells was reduced compared to that of the parental cell line.

/A A ' O r-^. I 1 i i - -i

TIME(min) Chart 2. Cellular uptake of [3H]pyridoxine into Fu5-5 and CI. 10 cells. [3H]- Pyridoxine (final concentration, 1.33 UM)was added to cell cultures as described in "Materials and Methods" and incubated for various times. At the end of each time point, the medium was removed, and the cells were washed 3 times in PBS and then dissolved in 1 N NaOH. Each time point represents the mean value of 3 T-75 sq cm flasks.

dose of pyridoxine for 5 to 6 weeks, colonies of cells appeared O 1 S 3 4 B which were then cloned. A cell line was established that was INCUBATION in PYRIDOXINE- resistant to 5 mM pyridoxine. This cell line is referred to as FREE MEDIUM(days) clone 10(01.10). Uptake of [3H]Pyridoxine into Fu5-5 and CI.10 Cells. Initial Chart 3. Uptake of [3H]pyridoxine into CI. 10 cells. Cells were initially plated in 5 mM pyridoxine-supplemented MEM and then changed to pyridoxine-free experiments to elucidate the mechanism of resistance of CI.10 MEM plus 5% dialyzed serum. Each day, cells were washed in serum-free, cells to 5 mM pyridoxine were centered on the uptake of [3H]- pyridoxine-free MEM and finally cultured in ttiis medium. [3H]Pyridoxine was added to each flask (final concentration, 1.33 JUM)and incubated for 30 min at pyridoxine into these cells. When both cell lines were analyzed 37Â°. Cells were washed 3 times in PBS and dissolved in 1 N NaOH. Results for their ability to take up [3H]pyridoxine, CI. 10 cells concen represent the mean value of 3 T-75 sq cm flasks.

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Fu5-5 cells had a population-doubling time of 12 hr compared Table 2 to a population-doubling time of 20 hr for CI.10R (data not Pi. and PLP content in Fu5-5 and CI. 10 cell lines The concentration of PL and PLP was measured in Fu5-5 and CI. 10 cells as shown). These results indicate that the cloned pyridoxine-re- described in "Materials and Methods." sistant cell line has properties distinct from the parental Fu5-5 cell line. Moreover, once established, CI. 10 cells retain their Concentration (nmol/mg protein) ability to adapt to high levels of extracellular pyridoxine. Cell line PLP PL Kinetics of [3H]Pyridoxine Uptake into Fu5-5 and CI. 10 0.328 Â±0.09a Fu5-5 0.116 Â±0.02 Cells. Saturation kinetics for the uptake of [3H]pyridoxine into CI.10 0.555 Â±0.04 0.172 Â±0.01 Fu5-5 and CI. 10 cells after 30 min of incubation is shown in Mean Â±S.D. of 4 individual T-75 flasks. Chart 4. A Lineweaver-Burke plot of the data generated 2 straight lines intersecting on the y-axis. Vma*values for the Fu5- 5 and CI.10 cells were 100 pmol per mg protein per 30 min. The apparent Km for the transport system in Fu5-5 cells was â€¢- 300- 0.71 /IM, and for the CI.10 cells it was 12.5 juM. Intracellular Levels of PL and PLP. It has been suggested that the intracellular accumulation of PLP can regulate its own O> formation (9). Since the inability of CI. 10 cells to concentrate [3H]pyridoxine was not due to an inoperative pyridoxal kinase enzyme (data not shown), the possibility existed that PLP could be expressing product inhibition of either pyridoxine oxidase or pyridoxal kinase. When the intracellular levels of PL and â€” PLP were measured, CI. 10 cells contained 69 and 48% more 0> PLP and PL, respectively, than did the Fu5-5 cells (Table 2). > 0>

Table 1 Growth rate of Fu5-5 and CI. 10R in 5 mM pyridoxine medium The effect of culturing Fu5-5 and CI.1 OR in 5 mm pyridoxine for 72 hr was determined by plating 1 x 106 cells in T-75 sq cm flask in the presence or FUS-S CI.IO CI. ICm FU5-5 CI.1OP absence of 5 mM pyridoxine. Cells were cultured as described in "Materials and Methods." Cell count was determined by the use of a hemocytometer in the SiiilVl BG Â« SmrVl BG presence of trypan blue. Results are the average of 3 T-75 sq cm flasks. rate(no. growth of cells/flaskx10")64.03.287.89.75 celi line CelllineFu5-5Fu5-5CI.1 Charts. Measurement of ATP levels in Fu5-5, CI.10, and CI.10R cells. Protocol for the experiment is described in "Materials and Methods." ATP was pyridoxineMEMMEM+ 5 mM measured in a luminometer M 1070 in the presence of the bioluminescence ORCI.1 reagent, LUMIT. ATP concentration is expressed as nmol/mg protein and rep ORMediaMEMMEM + 5 mM pyridoxine72-hr resents the means of 3 T-75 sq cm flasks.

o.s These high intracellular levels of PL and PLP could account for part or all of the reduced transport and subsequent trapping of [3H]pyridoxine in CI. 10 cells. Intracellular Levels of ATP in Fu5-5, CI.10, and CI.10R Cells. When the 3 cell lines were assayed for their intracellular ATP concentrations, it was found that Fu5-5 and CI.1 OR cells had approximately 215 nmol ATP per mg protein whereas the CI. 10 cells had ATP levels of 295 nmol ATP per mg protein (Chart 5). When the Fu5-5 and CI.1 OR cells were incubated for 24 hr in MEM supplemented with 5.0 mw pyridoxine and then assayed for ATP, the Fu5-5 cells had ATP levels of 210 nmol/ mg protein and the CI.1 OR cells had ATP levels of 277 nmol/ mg protein. These data indicate that CI. 10 cells had 27% more ATP than did the parental cell line. CI.1 OR cells had ATP levels equivalent to Fu5-5 cells when in MEM but in contrast to the parental cell line increased their ATP concentration when ex posed to MEM supplemented with 5 mw pyridoxine. Morphology of Fu5-5, CI.10, and CI.1 OR Cells. Initially, we thought that CI.10 cells might show cell surface changes that ^l/PHjPYRIDOXINE r1 could be correlated with vitamin B6 treatment. To investigate Chart 4. Lineweaver-Burke plot of the uptake of [3H]pyridoxine into Fu5-5 this possibility, 4 to 5 cultures each of Fu5-5 and CI.10 cells and CI. 10 cells. Cells were incubated with increasing concentrations of [3H] pyridoxine for 30 min at 37Â°. At the end of the incubation period, cells were were prepared for SEM. We were unable, however, to detect washed 3 times in PBS and then dissolved in 1 N NaOH. Results represent the any major observable differences in surface configuration be mean of 3 T-75 sq cm flasks. tween these 2 cell lines (Figs. 4 and 5).

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Our observation that ATP levels were increased in CI. 10 cells 5 cells were smaller than those of either CI. 10 or CI. 10R cells. suggested that pyridoxine might be affecting the mitochondrial Some pyridoxine-treated cells contained mitochondria that compartment. Thus, the fine structure of Fu5-5 and CI.10 cells were up to 3 to 4 times larger than those of the parental cell was examined after preparation for TEM. CI.10 cells appeared line. In Fu5-5 cells, the mitochondrial matrix was generally to contain a larger volume of mitochondria than did Fu5-5 cells dense (Fig. 1), while in CI. 10 and CI.10R cells it was typically (Figs. 1 and 2). To verify this qualitative observation, the pale and flocculent (Figs. 2 and 3). Cristae were relatively percentage of the cell volume occupied by mitochondria was sparse and were usually randomly oriented in mitochondria of determined by stereological methods for each of the cell lines. Fu5-5 cells. Although cristae were sparse and often randomly In Fu5-5 cells, 6.31% of the cell volume was occupied by arranged in mitochondria of pyridoxine-treated cells, they were mitochondria; whereas in CI.10 cells, this compartment consti also commonly aligned in rows perpendicular to the surface of tuted 9.92% of the fractional cell volume (Table 3). Therefore, the mitochondrion (Fig. 2). In addition, the CI. 10 and CI.10R the percentage of volume of mitochondria in CI. 10 cells was cells had an unusual configuration of cristae in which one or 50% greater than that of the parental cell line. Interestingly, more were closely packed and near to or hugging the edge of the mitochondrial volume of cells grown in pyridoxine-deficient the mitochondrion (Fig. 3, inset). Mitochondrial granules were medium resembled that of the parental cell line Fu5-5 (data not not prominent in any of the cell lines. All cultures examined shown). were prepared for microscopy at the same time, making it Biochemical measurements indicated that CI.10R cells cul unlikely that the observed differences in mitochondrial mor tured in MEM displayed levels of ATP similar to those of Fu5-5 phology were the result of variations in tissue processing. cells. CI.10R cells responded markedly to 5 rriM pyridoxine It is interesting that the changes seen in mitochondria of cells treatment by increasing their ATP levels. In contrast, the levels cultured in the presence of pyridoxine (CI. 10) persisted even of ATP in Fu5-5 cells remained essentially constant even when when these cells were subsequently cultured without this vita- they were exposed to high doses of the vitamin. Again, ster- mer (CI.10R). This implies that the alterations in mitochondrial eology was used to determine if the mitochondria of CI.10R structure brought about by pyridoxine treatment were of a cells were involved in the observed phenomenon. In CI.10R permanent nature. cells, mitochondria accounted for 10.11 % of the fractional cell volume. This value was virtually identical to that for CI. 10 cells DISCUSSION and represented a 50% increase over that for Fu5-5 cells, the parental cell line. We have recently shown that vitamin B6 may be a potential Analysis of variance indicated that there were significant antineoplastic agent (5). Data presented in that study clearly differences in the fractional cell volume of mitochondria among indicate that cells grown in a medium supplemented with 5 HIM the 3 cell lines (F = 36.4; d.f. = 2.11; p < 0.001). Although pyridoxine display signs of severe growth inhibition and ulti the means for the percentage of the cell occupied by mito mately are killed. However, the precise mechanism by which chondria in CI. 10 and CI.10R cells did not differ from each vitamin B6 retards the growth of cells was not ascertained. In other, that for Fu5-5 cells was significantly different from both the present communication, studies were undertaken in an CI. 10 and CI. 10R (p< 0.01). attempt to elucidate this mechanism(s). On the whole, the mitochondria of CI. 10 and CI.10R cells Our initial attempts to increase the intracellular levels of PLP also differed in morphology from those of the parental cell line, by culturing cells in high concentrations of pyridoxine resulted although there was some overlap in the appearance of this in either cell death or retardation of cell growth (Chart 1). It organelle among all cell lines. Overall, the mitochondria of Fu5- was necessary to adjust culture conditions to diminish toxicity

Table 3 Percentage ot cell volume occupied by mitochondria in Fu5-5. CI. ) 0. and CI. 10R cells

over of cell volume % of cell over other organ- occupied by mi volume occupied of varia CelllineFu5-5CI. mitochondria108107 elles1620b1618 tochondria6.256.20bymitochondria6.31 tion7.011.41.6

Â±0.44C9.92 3412 101120164194 1624160815461488 5.856.949.5911.53

10CI.10RSampleIa2 Â±1.13d10.11 3412 167153171176176174Points1558156815571542 9.678.899.8910.24

Â±0.16Coefficient 34Points 15451554% 10.2310.07Mean

" Each number designates an individual culture. 6 The fractional cell volume for mitochondria was determined by stereological methods as described in 'Materials and Methods." The maximum number of test points for 16 cells is 1728. However, the actual number may be smaller since points falling over extracellular space are not tabulated. c Average Â±S.D. of values in Column 5. The mean for Fu5-5 is significantly different from those for both CI.10 and CI.10R (p < 0.01). " The means for CI. 10 and CI. 10R do not differ significantly

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1982 American Association for Cancer Research. Pyridoxine Resistance in Hepatoma Cells to the cells. This was accomplished by increasing the concen high hexokinase activity (2) which is associated with the mito- trations of pyridoxine in the medium over a period of 3 to 4 chondrial fraction. Coupled to the elevated hexokinase activity months. Using this protocol, a cell line was established that are alterations in the glycolytic rate and changes in mitochon- was resistant to killing by 5 HIM pyridoxine. The experiments drial volume (3). presented in this communication describe this cell line (CI. 10) Two of our observations suggest the possible inhibition of and suggest its value for the study of the regulation of cell hexokinase by the increased levels of PLP in CI. 10 cells, (a) growth by vitamin B6. Pyridoxine treatment leads to a marked decrease in the growth The CI. 10 cell line may prove a valuable tool in other areas rate of CI.10 cells. The population-doubling time of CI.10 cells of research, such as studies of vitamin B6 metabolism. It has was 24 hr, while that of Fu5-5 cells was 12 hr. The slower been suggested by Mehansho et al. (13) that pyridoxine is growth rate of CI. 10 cells could result if PLP was inhibiting passively transported into the cell and trapped by phosphory- glycolysis by blocking the activity of the glycolytic enzymes lation to pyridoxine 5'-phosphate, a reaction catalyzed by mentioned above, (o) We found that CI. 10 cells had both pyridoxal kinase. Kwok and Churchich (9) have shown that increased levefs of ATP and a 50% increase in the mitochon- both pyridoxal kinase and pyridoxine oxidase are involved in drial volume compared to the parental cell line. These findings the regulation of PLP, and Li ef al. (10) have shown, in vivo lend further support to our theory that PLP may inhibit the and in vitro, that the control mechanisms regulating the cellular activity of hexokinase, with the consequent slowing of the content of PLP operate even when vitamin B6 is present in glycolytic rate. Although conclusive evidence is lacking, addi excess. We have shown here that it is possible to increase the tional support for our hypothesis comes from the work of intracellular concentration of PLP by culturing cells in an ex Jayaraman ef al. (8). In their studies, high levels of glucose cess of vitamin B6. Kinetic studies revealed that Vmaxfor the suppressed the formation of mitochondria, whereas when the uptake of [3H]pyridoxine in CI. 10 cells was the same as in the concentration of this sugar was reduced functional mitochon parental line (Chart 4). However, the apparent Km for the dria were formed. transport system in CI.10 cells was 12.5 JUM,almost 18 times Although we have not described the exact molecular mech higher than for Fu5-5 cells. Since both cell lines had approxi anism of pyridoxine resistance, the newly established cell line mately the same level of pyridoxal kinase activity (data not (CI. 10) has 69% more intracellular PLP, 27% more intracellular shown), it is possible that the reduced uptake of [3H]pyridoxine ATP, and a 50% increase in the volume of the cell occupied by into CI.10 cells was the result of product inhibition by either mitochondria as compared to the parental cell line (Fu5-5). PLP or PL or both on the enzymes involved in the intracellular These parameters ultimately may be involved in the mechanism trapping of pyridoxine. of resistance. Also, it is conceivable that pyridoxine resistance However, we realize that other factors may be contributing may be traceable to a genetic alteration, providing an additional to the reduced cellular uptake of [3H]pyridoxine. Since we have marker in somatic cell genetics. not investigated the intracellular metabolism of PLP, it may be possible that the enzymes responsible for the synthesis and/ REFERENCES or degradation of PLP have been altered or inhibited to some 1. Bornstein. M. B. Reconstituted rat-tail collagen used as a substrate for tissue degree resulting in an accumulation of PLP metabolites. In the culture on cover slips in Maximow slides and roller tubes. Lab. Invest.. 7: present study, only intracellular PL and PLP were measured. 134-137, 1958. Therefore, it is possible that other vitamine B6 metabolites 2. Bustamante, E., Morris, H. P.. and Pederson. P. L. Energy metabolism of tumor cells. Requirement for a form of hexokinase with a propensity for (pyridoxamine, pyridoxamine phosphate, pyridoxine phos mitochondrial binding. J. Biol. Chem., 256. 8699-8704, 1981. phate) could play a role in the regulation of the cellular uptake 3. Bustamante, E., and Pederson, P. L. High aerobic glycolysis of rat hepatoma of [3H]pyridoxine. Another factor which merits consideration is cells in culture: Role of mitochondrial hexokinase. Proc. Nat. Acad. Sei. U. S. A. 74. 3735-3739, 1977. the possibility that the total intracellular protein concentration 4. Deschatrette, J.. and Weiss, M. C. Characterization of differentiated and could be reduced in CI.10 cells thus affecting not only the dedifferentiated clones of a rat hepatoma. Biochimie (Paris), 56. 1603- amount of PLP bound to protein but also the osmotic activity of 1611, 1974. 5. DiSorbo, D. M., and Litwack, G. Vitamin B6 kills hepatoma cells in culture. the cell. Therefore, it is clear that more intensive investigation Nutr. Cancer, in press. 1982. must be carried out on CI. 10 cells before definitive conclusions 6. Farquhar. M. G., and Palade, G. E. Cell junctions in amphibian skin. J. Cell Biol., 26. 263-291, 1965. can be drawn concerning vitamin B6 uptake and metabolism in 7. Glazer, B. I., and Weber, G. Pyridoxal 5'-phosphate inhibition of lactate this cell line. Nevertheless, it is important to bear in mind that production in liver supernatant fluid. Biochim. Biophys. Acta, 237. 11-13, the defective pyridoxine uptake developed in CI. 10 cells can 1971. 8. Jayaraman, J., Gotman, C., Mahler. H. R., and Sharp, C. W. Biochemical be reverted to a level approaching that of the parental cell by correlates of respiratory deficiency. VII. Glucose repression. Arch. Biochem. removal of pyridoxine from the medium, all this while the Biophys., 116. 224-251, 1966. apparent mutation of resistance to killing is retained. 9. Kwok. F.. and Churchich, J. E. Interaction between pyridoxal kinase and pyridoxine-5-P oxidase, two enzymes involved in the metabolism of vitamin Another point that merits discussion is the possible role of B6. J. Biol. Chem., 255. 882-887, 1980. PLP in the regulation of glycolysis. Srivastava and Beutler (18) 10. Li, T-K., Lumeng, L., and Veitch, R. L. Regulation of pyridoxal 5'-phosphate have shown that, in RBC, PLP inhibits hexokinase, phospho- metabolism in liver. Biochem. Biophys. Res. Commun., 61: 677-684, 1974. 11. Lowry, O. H.. Rosebrough. N. J.. Farr. A. L.. and Randall, R. J. Protein fructokinase, and pyruvate kinase, 3 enzymes involved in gly measurement with the Folin phenol reagent. J. Biol. Chem., )93: 265-275. colysis. PLP has also been shown to inhibit lactate production 1951. 12. Masurovsky. E. B., and Bunde. R. P. Fluoroplastic coverslips for longterm in rat liver supernatant fluid, possibly through the inhibition of nerve tissue culture. Stain Technol., 43: 161-165, 1968. hexokinase (7). 13. Mehansho. H., Buss, D. D.. Hamm, M W., and Henderson. L. M. Transport The relevance of PLP inhibition of hexokinase to the present and metabolism of pyridoxine in rat liver. Biochim. Biophys. Acta, 637. 112- 123, 1980. work can be best appreciated when related to other available 14. Paavola. L. G.. and Boyd, C. O. Cytoplasmic granules in luteal cells of information. Hepatoma cells characteristically have abnormally pregnant and non-pregnant guinea pigs. A cytochemical study. Anat. Ree.,

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201: 127-140, 1981. on the activities of red cell enzymes. Arch. Biochem. Biophys., 148: 249- 15. Paavola, L. G., and Christensen, A. K. Characterization of granule types in 255, 1972. luteal cells of sheep at the time of maximum progesterone secretion. Biol. 19. Wada, H., and Snell, E. E. The enzymatic oxidation of pyridoxine and Reprod., 25. 203-215, 1981. pyridoxamine phosphates. J. Biol. Chem., 236: 2089-2095, 1961. 16. Reynolds, E. S. The use of lead citrate at high pH as an electron opaque 20. Weinstein, A., Medes. G., and Litwack, G. Isotopie method for tyrosine stain in electron microscopy. J. Cell Biol., 1 7: 208, 1963. transaminase activity. Anal. Biochem., 21: 86-97, 1967. 17. Spurr, A. R. A low-viscosity epoxy resin embedding medium for electron 21. Williams, M. A. Stereological techniques. In: A. M. Glauert (ed.). Practical microscopy. J. Ultrastruct. Res.. 26. 31-43, 1969. Methods in Electron Microscopy, Vol. 6. pp. 5-84. New York: North-Holland 18. Srivastava, S. K., and Beutler, E. The effects of normal red cell constituents Publishing Co., 1977.

2368 CANCER RESEARCH VOL. 42

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Fig. 1. TEM of Fu5-5 cell cultured Â¡nMEM. Note that the mitochondria (m) of Fu5-5 cells are modest in size, have a dense matrix, and contain relatively sparse, randomly oriented cristae. er, rough endoplasmic reticulum; n, nucleus. Uranyl acetate and lead citrate, x 15.000. Fig. 2. TEM of CI. 10 cells grown in MEM containing 5 mw pyridoxine. Note that, in contrast to Fu5-5 cells, the mitochondria (m) of CI. 10 cells are large and contain a pale, flocculent matrix, er, rough endoplasmic reticulum; n, nucleus, x 15,000. Inset, many mitochondria of pyridoxine-treated cells containing cristae (arrowheads) that are aligned in parallel rows. Uranyl acetate and lead citrate, x 27.800.

JUNE 1982 2369

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Fig. 3. TEM of CM OR cell cultured in MEM. Note that, even in the absence of high concentrations (mM) of pyridoxine in the medium, mitochondria (m) of CI.10R cells do not revert to the morphology typical of the parental cell line. Fu5-5. Instead, they resemble the mitochondria of CI. 10 cells; i.e., they are large with a pale, flocculent matrix, er. rough endoplasmic reticulum; n, nucleus, x 15.700. Cristae in mitochondria of both CI.10 and CI.10R cells may have an unusual arrangement; one (arrowheads) or more cristae hug the edge of the mitochondrion (inset). Uranyl acetate and lead citrate, x 27,800. Fig. 4. Scanning electron micrographs of Fu5-5 cells, showing their typical surface morphology. The surfaces of Fu5-5 cells are studded with many microvilli which vary in length, x 1800. Fig. 5. Scanning electron micrographs of CI. 10 cells, showing their typical surface morphology. Note that the surfaces of CI. 10 cells do not differ markedly from those of the parental cell line Fu5-5, although the microvilli of CI. 10 cells tend to be short and of relatively uniform length, x 1800

2370 CANCER RESEARCH VOL. 42

Dennis M. DiSorbo, Laurie G. Paavola and Gerald Litwack

Cancer Res 1982;42:2362-2370.

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