The Effect of Terramycin on the Fine...
16
The Effect of Terramycin on the Fine Structure structure of HeLa cell mitochondria by the anti biotic terramycin. MATERIALS AND METHODS HeLa cells were grown on standard 3 â€oeXI― micro slides in square French bottles for bright field and UV studies. Control cultures and experi mental cultures growing in varying concentrations of terramycin were rinsed in Hanks solution and mounted in control medium under coverslips sealed with paraffin and observed immediately with a Leitz fluorescent unit. Fixed cultures were stained by the aniline-acid fuchsin-@methyl green method or the phosphotungstic acid-hematoxylin procedure for mitochondria (15). For electron microscopy, stock HeLa cells were grown in a medium composed of 1 part calf serum and 2 parts medium 199 in stationary roller tubes. After monolayers had developed, terramycin (Pfizer brand of oxytetracycline) was added to the tubes in concentrations ranging from 100 to 1000 j@g/ml culture medium. The pH of the media con taming terramycin ranged between 7.1 and 7.4. After exposure to the antibiotic for 2—24hours the cells were fixed in sAtu by exposure for 10 minutes to vapors from a 2 per cent osmic acid. solution. Embedding was carried out in methacrylate as pre viously described (11). Thin sections were mount ed on formvar films and stained with lead hydrox ide (21) before examination. The tetracycline series of antibiotics is used in the treatment of bacterial infections in man and laboratory animals. In the laboratory, terramycin, one of the tetracyclines, is frequently added to tis sue culture media to prevent the growth of bacteria and PPLO organisms. As with most antibiotics, the exact mechanisms of bactericidal action of the tetracyclines are unknown. It is, however, becom ing increasingly apparent that they may also dam age the cells of higher organisms. Earlier reports have indicated that certain antibiotics have dele terious effects on the growth and functions of cells in culture (4, 10, 14, 19); however, the degree of toxicity for bacteria is usually greater than it is for the mammalian cell. Terramycin is readily detected in animal tissues soon after injection because of its brilliant yellow green fluorescence (20, 28). Recent studies by DuBuy and Showacre (6) indicated that the tetra cyclines were rapidly incorporated into cells, both in vivo and in vitro, and that these antibiotics were localized in the mitochondria. Biochemical studies indicated that the antibiotics caused a decrease in oxidative phosphorylation when added to isolated mitochondria (6). The present study is concerned with the specific alterations produced in the fine Supported by grant #527 from the Damon Runyon Fund for Cancer Research. t Present address: St. Jude Hospital, Memphis, Tennessee. Received for publication October 3, 1962. 5.51 . of HeLa Cell Mitochondria* L. J. JOURNEYANDMILTON N. GOLDSTEINt (Department of Experimental Biotogij, Roawell Park Memorial InsiiMe, Buffalo, N.Y.) SUMMARY Terramycin, in various concentrations, was added to culture media in which HeLa cells were proliferating. Bright-field and fluorescent microscopy revealed that terra mycin is rapidly and specifically bound to the mitochondria. At low concentrations no changes in mitochondrial morphology were apparent. When concentrations were in creased to 100 @g/mlor higher, the mitochondria became distended and rounded up. Marked alterations in mitochondrial structure were observed at concentrations of 100 @g/ml with the electron microscope. These changes included vacuolation, corn pression of the cristae, and striking reduplication of the mitochondrial membranes. At 300 @g/mlno normal mitochondria were present, and degeneration of other cytoplas mic elements occurred. Concentrations of 1000 /hg/ml proved lethal for all cells. on March 19, 2019. © 1963 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
-
Upload
truongdung -
Category
Documents
-
view
220 -
download
0
Transcript of The Effect of Terramycin on the Fine...
The Effect of Terramycin on the Fine Structure
structure of HeLa cell mitochondria by the antibiotic terramycin.
MATERIALS AND METHODSHeLa cells were grown on standard 3 “XI―
micro slides in square French bottles for brightfield and UV studies. Control cultures and experimental cultures growing in varying concentrationsof terramycin were rinsed in Hanks solution andmounted in control medium under coverslipssealed with paraffin and observed immediatelywith a Leitz fluorescent unit. Fixed cultures werestained by the aniline-acid fuchsin-@methyl greenmethod or the phosphotungstic acid-hematoxylinprocedure for mitochondria (15).
For electron microscopy, stock HeLa cells weregrown in a medium composed of 1 part calf serumand 2 parts medium 199 in stationary roller tubes.After monolayers had developed, terramycin(Pfizer brand of oxytetracycline) was added to thetubes in concentrations ranging from 100 to 1000j@g/ml culture medium. The pH of the media contaming terramycin ranged between 7.1 and 7.4.After exposure to the antibiotic for 2—24hours thecells were fixed in sAtu by exposure for 10 minutesto vapors from a 2 per cent osmic acid. solution.Embedding was carried out in methacrylate as previously described (11). Thin sections were mounted on formvar films and stained with lead hydroxide (21) before examination.
The tetracycline series of antibiotics is used inthe treatment of bacterial infections in man andlaboratory animals. In the laboratory, terramycin,one of the tetracyclines, is frequently added to tissue culture media to prevent the growth of bacteriaand PPLO organisms. As with most antibiotics,the exact mechanisms of bactericidal action of thetetracyclines are unknown. It is, however, becoming increasingly apparent that they may also damage the cells of higher organisms. Earlier reportshave indicated that certain antibiotics have deleterious effects on the growth and functions of cellsin culture (4, 10, 14, 19); however, the degree oftoxicity for bacteria is usually greater than it is forthe mammalian cell.
Terramycin is readily detected in animal tissuessoon after injection because of its brilliant yellowgreen fluorescence (20, 28). Recent studies byDuBuy and Showacre (6) indicated that the tetracyclines were rapidly incorporated into cells, bothin vivo and in vitro, and that these antibiotics werelocalized in the mitochondria. Biochemical studiesindicated that the antibiotics caused a decrease inoxidative phosphorylation when added to isolatedmitochondria (6). The present study is concernedwith the specific alterations produced in the fine
Supported by grant #527 from the Damon Runyon Fundfor Cancer Research.
t Present address: St. Jude Hospital, Memphis,Tennessee.
Received for publication October 3, 1962.
5.51
. of HeLa Cell Mitochondria*
L. J. JOURNEYANDMILTON N. GOLDSTEINt
(Department of Experimental Biotogij, Roawell Park Memorial InsiiMe, Buffalo, N.Y.)
SUMMARYTerramycin, in various concentrations, was added to culture media in which HeLa
cells were proliferating. Bright-field and fluorescent microscopy revealed that terramycin is rapidly and specifically bound to the mitochondria. At low concentrations nochanges in mitochondrial morphology were apparent. When concentrations were increased to 100 @g/mlor higher, the mitochondria became distended and rounded up.
Marked alterations in mitochondrial structure were observed at concentrations of100 @g/ml with the electron microscope. These changes included vacuolation, corn
pression of the cristae, and striking reduplication of the mitochondrial membranes. At300 @g/mlno normal mitochondria were present, and degeneration of other cytoplasmic elements occurred. Concentrations of 1000 /hg/ml proved lethal for all cells.
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
552 Cancer Research Vol. 23, May 1963
RESULTS
Bright-field microscopy.—The mitochondria inHeLa cells grown for 24—48hours in media containing 5—10,@g/ml of terramycin were morphologicalIy like those in cells grown in control media. Fixedand stained preparations contained many long,filamentous mitochondria. When the concentration of the antibiotic was increased to 100 ,@g/ml,a shortening and swelling of the mitochondria occurred after 8—10hours of incubation. Some cellsalso contained small numbers of spherical mitochondria. If the concentration of the antibiotic wasraised to 300 @zg/mlor higher, marked alterationsin the morphology of the mitochondria occurredwithin 2 hours of incubation. The mitochondriadistended and became spherical in shape. On continued incubation at these higher concentrations,no normal-appearing mitochondria were seen after6 hours. Cell lysis was observed as early as 10 hoursafter the addition of these high concentrations ofantibiotic, and all the cells appeared granular.Most of the cells had lysed after 24 hours' incubation.
Uv fluorescence.—Fiveminutes after the addition of 5 j.ig/ml of terramycin the mitochondriafluoresced bright green. At this, or slightly higher,concentration there were no alterations in themorphology of the mitochondria as observed in thefluorescent microscope. Morphological alterationssimilar to those in fixed sections were seen whenthe concentration of the antibiotic was increasedto 100 ,@g/ml or higher. At all the concentrationsfluorescence was limited to the mitochondrial elements.
Electron microscopy.—The fine structure ofHeLa cells has been described previously (11). Themitochondria of HeLa cells vary considerably inmorphology, ranging from short rods to long, filamentous structures@ (Fig. 1). Cristae often appeared irregularly oriented and were not abundantin the filamentous variety. The matrix had a relatively low electron density. These observationshave been confirmed by other investigators (@,7).
After exposure to 100 @gterramycin/ml for 1%hours all the cell organelles appeared as they didin control cultures except for the mitochondria.Although the structure of most mitochondria wasdefinitely altered, a few appeared unaffected bythe drug. The degree of mitochondrial alterationsvaried somewhat from cell to cell and may havedepended upon the metabolic state of the cells.Slight damage was indicated by a localized distention or vesiculation. Many of the mitochondriawere ring-shaped, probably representing horizontal sections through cup-shaped structures. Somemitochondria were rounded, with characteristic
cristae displaced by stacks of membranes (Fig. 2).At 200 @g/mldamaged and altered mitochon
dna were observed in the electron microscope incells which had been incubated for as little as ‘2hours. These damaged mitochondria appearedswollen; some retained a disorganized mass of internal membranes (Fig. 3), and other organellescontained a granular matrix enclosing heavywalled vesicles (Fig. 4).
At 300 ,@g/ml,mitochondrial damage was further accentuated; some of the mitochondria consisted of complex membranous whorls, and othershad dense myelin-like inclusions located within thevacuoles. Some of these organelles were enormously swollen. In several instances the cristae werecompressed toward one end of the mitochondria;in others the cristae appeared to have been replacedby dense granular material (Figs. 5, 6). At thisstage, vacuolation of the endoplasmic reticulumwas evident in many cells.
In later stages of degeneration, many of themitochondria were transformed into large vesicleswith translucent interiors surrounded by a thickperiphery. Others retained their multivesicularnature, with vacuolar contents in various stages ofdegeneration. The nuclear structures appearednormal, but other signs of cell necrosis becomeapparent. There was a condensation of alteredmitochondria and other cytoplasmic elements,which were interspersed with a fibrous materialof unknown origin. Hyalinization of the cytoplasm at the cell periphery was also a noticeablefeature (Fig. 7).
The effects of terramycin appear to be primarilyevident in mitochondria. At a concentration of theantibiotic (100 ,@g/ml) at which marked changesin mitochondrial morphology were noted, othercytoplasmic structures did not appear to be altered. There was no evidence of nuclear changesor vacuolation of the endoplasmic reticulum. Atthe higher concentrations, evidence of cytoplasmicdegeneration was widespread after 10—12hours ofincubation. These changes may, however, reflectsecondary effects in cells deprived of normal mitochondrial function for extended periods of time.
DISCUSSIONStudies by other investigators have demon
strated that the tetracycline antibiotics are preferentially bound to mitochondria (6). Our experiments have shown that terramycin, ox@ytetracycline,in concentrations of 100 ,@g/ml or higher, rapidlyproduced striking alterations in the morphologyof mitochondria in HeLa cells. The initial reactioninvolved vacuolation of the mitochondria, followed by compression of the cristae and/or redu
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
JOURNEY AND GOLDSTEIN—Effect of Terramycin on Mitochondria 553
plication of the mitochondrial membranes to formmultilaminar bodies. As degeneration progressed,these changes were accentuated, the characteristiccristae were lost, and the organelles were converted to thick-walled vacuoles or myelin-like figures.At higher concentrations of the antibiotic, degeneration of the mitochondria was followed by celllysis.
Mitochondrial alterations, similar to those described above, were among the structural changesseen in L cells following vaccinia virus infection(5), and adenoviruses have been reported to stimu
late the reduplication of perinuclear membranes(9). Transformation of mitochondria into lamellar
bodies has been described in epidermal (18) andHeLa cells (13),when the composition of the tissueculture media was varied. Cup-shaped mitochondrial profiles were observed occasionally in rat testicular interstitial cells (3). Formation of concentric lamellae has also been seen in inclusion bodiesof alveolar phagocytes following ingestion of Indiaink (12).
In a rapidly multiplying population of cells asmall percentage of the cells undergo degeneration.Morgan et at. (22) have observed vacuolation andcompression of cristae in mitochondria, vesiculation of the endoplasmic reticulum, and, in moreadvanced stages, condensation of the cytoplasmiccontents during spontaneous necrosis of culturedcells.
With regard to biochemical lesions produced bytetracyclines, several reports have noted that oxidative phosphorylation is particularly sensitive tothese antibiotics. Loomis (16) first noted thataureomycin specifically depressed phosphorylationwithout inhibiting respiration of mitochondria.DuBuy et at. (6) found oxygen consumption unchanged but a decreased efficiency of oxidativephosphorylation in the presence of tetracyclineand terramycin as well as aureomycin. Brody et a!.(1) studied a series of tetracyclines and concludedthat in vitro inhibition of oxidative phosphorylation may be due to the chelating ability of thetetracyclines. They postulated that tetracyclinescombine with and effectively remove Mg@, whichis necessary for the coupling reaction to proceed.If the binding of terramycin to cell structures alsodepends on its chelating ability, then its preferential localization in mitochondria would appear toindicate binding with the Fe@ of the cytochromes.
From extensive studies of fractionated mitochondria, Green (8) found that the double-membrane structure is necessary for coupling oxidationto phosphorylation and stated that the respiratoryenzymes are rigidly organized and integrated within the architecture of the mitochondria. Because of
this close interrelationship of structure and function in mitochondria, it is difficult to determineexactly where terramycin has its effect The initialeffect may be on the enzymatic machinery, and theconsequent loss of respiratory activity would leadto degeneration of mitochondrial organization.Inhibition of the energy-producing mechanisms ofthe mitochondria then would block the metabolicprocesses of the cell involved in carbohydratemetabolism and eventually lead to cell destruction.It is less probable that the tetracyclines directlymodify mitochondrial structure upon which enzymatic activity depends, thus resulting in celldestruction.
The addition of as little as 5 jig/ml of terramycm to our tissue culture mediumresulted in thefluorescence of the mitochondria of living HeLacells which were exposed to UV light. Since higherconcentrations are often used in tissue culturemedia, one may presume that levels below thoseneeded to produce cytotoxic effects may interferewith the “normal―function of the mitochondria.In some instances, cells used for measuring metabolic activities were grown in media containingfour different antibiotics (17). With the possibleexception of penicillin, which is apparently nontoxic to growing cells even at high concentrations,it appears that most antibiotics have specific effects on the host cell as well as infecting bacteria.One should, therefore, be cognizant of these inhibitory effects when interpreting data on metabolic rates derived from cells grown in mediacontaining antibiotics.
REFERENCES
1. BRODY,T. M.; Himwrrz, R.; and BAIN,J. A. Magnesiumand the Effect of the Tetracycline Antibiotics on OxidativeProcesses in Mitochondria. Antibiotics & Chemotherapy,4:864—70,1954.
2. BRUNI, C.; GET, M. K.; and Svo@nus, M. Changes in theFine Structure of HeLa Cells in Relation to Growth. Bull.Johns Hopkins Hosp., 109:160—78, 1961.
S. CHRISTENSEN, A. K., and Cu.apatax, G. B. Cup-shapedMitochondria in Interstitial Cells of the Albino Rat Testis.Exp. Cell Res., 18:576—79, 1960.
4. CRUICKSIIANK,C. N. D., and LOWBUBY,E. J. L. Effects ofAntibiotics on Tissue Culture of Human Skin. Brit. Med.J.. 2:1070—72,1952.
5. DALES,S., and Sn&n@ovrrcn,L. The Development of Vaccinia Virus in Earle's L Strain as Examined by ElectronMicroscopy.J.Biophys. Biochem Cytol., 10:475—SOS,1961.
6. DuBuy, H. G., and SHOWACRE,J. L. Selective Localizationof Tetracycline in Mitochondria of Living CeUs. Science,133:196,1961.
7. EPSTEIN, M. A. Some Unusual Features of Fine StructureObserved in HeLa Cells. J. Biophys. Biochem. Cytol., 10:158—63,1961.
8. GREEN, D. E. Mitochondrial Structure and Function.Laboratory Invest., 8:448—59,1959.
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
Cancer Research Vol. 23, May 1963554
9. GREGG,M. B., and MORGAN,C. Reduplication of NuclearMembranes in HeLa Cells Infected with Adenovirus. J.Biophys. Biochem. Cytol., 6:539-40, 1959.
10. Hu, F.; Lxvniooon, C. S.; JOHNSON, P.; and PoMnaaf,
C. M@Tissue Culture Studies @iHuman Skin. J. Invest.Dermatol., 20:857—72, 1953.
11. JoURNEY, L. J., and Gou,ørw@, M. N. Electron Microseope Studies on HeLa Cell Lines Sensitive and Resistantto ActinomycinD. Cancer lIes., 21:929-32, 1961.
12. KaRi@aR,H. E. ElectronMicroecopicStudyof the Phagocytic Process in Lung. J. Biophys. Biochem. [email protected]:857-€7,1960.
13.Kojmu, L, and KOZUKA, S.StructuralChanges inHeLaCellsCultivated in Serum Free Medium. J. CellBioL,14:141-44, 106%.
14. Lunra, P.; Bassszi, G.;aad MAuazsi,J. Action of Chioromycetin and Aureomycin on Normal Tissue Cultures.Proc. Soc. Exp. Biol. Med., 73:252-55, 1050.
15. LnUE, R. D. HistopathologicTechniqueand PracticalHistochemistry, pp. 183-85. New York: Blakiston Co.,1057.
16. LooMis,W. F. On the Mechanismof Actionof Aureomycm. Science,111:474, 1950.
17. MaDDEN, R. E., and Btrax, D. Production of Viable SingleCellSuspensionsfrom SolidTumors. J. Natl. Cancer Inst.,27:841—55,1061.
18. MaNurER, M. G., and EVANs,V. J. Structural DifferencesProduced in Mammalian Cells by Changes in Their Environment. J. NatL Cancer Inst., 25: 1303-25, 1960.
19. Murzonu, J. F.; Fusm@o, M. H.; CORNMAN,I.; andKUMNS, D. M. Antibiotics in Tissue Culture. Exp. CellRes.,6@S37-44, 1954.
20. MucH, R. A.; Ras@, D. P.; and ToBlu, J. E. Bone Localization of the Tetracycines. J. Nati. Cancer Inst., 19:87-92, 1957.
21. MILLONIG,G. A ModifiedProcedurefor LeadStainingofThin Sections. 3. Biophys. Biochem. Cytol., 11:729-83,1961.
22. Monoasi, C.; RosE, H. M.; and Mooau, D. H. An Evaluation of Host Cell Changes Accompanying Viral Multipliestion as Observedwith the Electron Microscope.Ann. N.Y.Acad. Sd., 68:802—23, 1957.
23. RAu@, D. P.; Loo, T. L.; L@u, M.; and KELLY, M. G.Appearance and Persistence of Fluorescent Material inTumor Tissue after Tetracycline Administration. J. Nati.Cancer Inst., 19:79—85, 1957.
FiG. L—Coatml HeLa cell. Part of the nucleus (suc) appears at lower left, and the cell periphery with a few microvilli
@ourseaalong the top of the micrograph. Hyallne central areawhich consists of a network of filaments corresponds to glycogen (clii). The irregularly arranged cri@tae are evident in themitochondriascatteredthroughthecytoplasm.X22,000.
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
1/ J : • :.@@@@ C:?;@@ .@@@ .T@ ‘@ •‘ .
@4@'.-:@ â€., ..,.;@ .. . @t.- .-. .,@ ..,.s. •,@. . . . ‘I@@ .@..:@ ‘t@
4@ c @. t
@4. - @a..' .. -. ..@, -. .. ,@ • @-@ .@ @.S@ • . •@ .. ,‘. . ‘S@ .@I@
.* ,@ &. -#@@ I@@@
@ a.. ‘ •@. f : :@ • .. .-. ..@ . ‘. 7@ .@‘ •
. .. 4 •@.p(_@@ a@—.:. .@@@ ‘@ @-: p@ . .- ti . . .@ A;,@@@ o@@ ..,. . .@ ?;t@ ;‘ .@@@ -,,_;j,.. ; .. 3)j@ ‘p • @, •@ ‘@@@ t
@. .v,,..@ @‘@‘:@@ ‘-V.
- I .4@Of.@! .
@,@ ,. I..:@@ - - . .@
.4- . - .@@ ‘-@.. ‘,
_____ .@. .@:* .@ ...- ‘-
• - -• . • ‘.‘—-
—‘.,@ . - . ..
g@
-;@ /@!
ii -‘
- I @*,“
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
FIG. @2.—Portions of three cells from a culture exposed to100 @igterramycin/ml for 1@hours. Nucleus (nuc) of one cellappears at upper right. Alterations in mitochondrial structureare readily apparent. These changes include localized vesiculation (1), ring-shaped forms (2), and multiplication of membranes with compression of the cristae (3). Several mitochondria in this field still retain normal structure. X2@,000.
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
• - çv'@ .@ • /
- - @l%@_ ‘•@
• . •--•@ ‘. @—•-• @, •
::@ ‘@::@:@@
. - : -@
4 ‘ -, da:.••,..@ - :@ -•
@ ;.@@ ;t@ ‘@@ *
.‘ • - ii@@@ -•‘@1@-, -- . ••@ ‘!@•@
@ -@@ T@10J : ‘@@ .@ ;@*(@ ,..@ r@
‘@@ ‘@‘@@@@ V4,511@
@4@@@@ ,@ @:‘@@ I ::@ ::a:@
—@@ 4• @@‘L@(@ @: ‘V
‘ ltf@ @A4 .. ‘@ c@ 4@ -4@ V .@@
V@ @J@ .@ @• @- ‘@-@ -..@
,@ _ @- J@t@ •,@* \@ 4@―@-:
-,:@ •@•@ 4@- .,:@
:.@ “@ t@;:@¼@:@.
@:@4
I'@
•p.
-•@I@
•, ,
•@
r /@ .ç
4 j@'
@•@@ 4@i•@@ I
.•.@J••J
••
,@ S
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
FiG. 3.—Part of a HeLa cell from a culture grown in @00 @gterramycin/ml for 2 hours. This section contains examples ofmitochondria with a localized vesiculation (1), a ringlike shape(2), and a swollenorganellein whichthe cristae are present asa tangled network (3). X@2,000.
Fio. 4.—Another example of mitochondrial damage produced in cells grown in media containing @00pg terramycin/mlfor 2 hours. Cristae have disappeared from these mitochondriawhich have a granular matrix and thick-walled vesicles (1), oneof which contains a large vacuole (2). X 16,000.
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
.#@ p,. ‘5
.- ;@@ ‘@@“@Z
;_., S
@-.
,_* 5- 4 - •‘.w:@ —WA@ L@
:@ S h' , ‘@ ‘@‘@ ; @.•,;-,,‘@ ;@
,@1' .@‘@,†@cvr@ ...s@@/―@@ ,@.-@‘VI:;‘@f -.@@ !cI@@.4@h.;;---.-@ :.@ ‘@@ q4@@“@ :@: I@@‘@ •@- @11@fr@;c4@ C
@ S. ‘@:4: :@p*@@.:@r::,4s%@ ..v
;@- ___•;@@.@@ ;@•Ii:@@@@@ -,@ d?L;@:@:
.&i ta.@ •.s-.-!@@ ‘-4_@__1
@@ •@-@ •@‘.@-•@@@ .
,u;@ ‘@@ ‘ ‘ •. £@ - s@‘@- @‘@-@@ @‘?@ , . -*4@_@@@ •@ @-,@ _,@ : @.@ L@@‘-@@@ ‘@ ‘I•@@ - : .
@@ ..-. -@ S@@ ,VS.-@ ?@: •@ ••• S@@@ .- @_:@ -@0@ •@@ . @V- -@ •@.3@p•@z7@:@
‘@{J/4:5*_ ‘V@ 4@@ @@4IIrr.Ir@@
@ @. I@ @- .,,.•‘ ,-@ @t ‘@c @‘@
c-V, .@@ - o •@@
‘ @-@ s@ @:,@
J,. -@@@ @4--i-@ I@ ‘-.@•.@j @—•@ @_: -@
@ d@
.- • ‘@ •@ “-C@@@@ -4':-@-: •@1@S@
.@@ @@•‘t-@―. .- , .•. -@ -- ,..c —
..- ‘@@ @‘•:@@ ‘@ %‘_‘_.,,_ •t@3
:@@?@@ :@
:‘.-
-,@-
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
FIG. 5.—Micrograph of HeLa cells after exposure to 300 pgterramycin/mi for 24 hours. The nucleus (nuc) appears atlower left. Mitochondrial destruction is widespread. Some appear as multilaminar bodies (1), others are vacuolated andcontain dense granular material (2). Vesiculation of the endoplasmic reticulum becomes evident (3). Few, if any, normalmitochondria are present at this concentration of terramycin.X28,000.
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
V@ @‘- •@ -@@ -;@@-@p- ,,@1k
1@ - @J@V@i@';@4@:@ •@. @-
@ ‘@‘V@p a@'c'@@ ;@ -S .@ - “4
s@f &@ —@r(@3I)@ £@ 41@q( o@@ @r.C*I@ L@ 1;'@@@
L@ ,@ ,‘@._@:;@@ d@@@- @FP;@t .,,‘ [email protected]. •T •@•@ @- . Si-@ •f@
,4@rT:l@ ,, @1:@-@lr.@ •.@‘E:@p I /T:'@ as
I'@ hr@ ‘ *r@@@@ ‘ ,+@@ .@b@V@ 4
—@ ..-@@ .-@ :, •@-‘*e.@.• ‘@ 4 .-@:@ -@ @P@\@ ‘>‘@‘@@‘‘@ 4@ib@'@ 4@@@ @.5,t.@ - •-4_.r@ ,Y@.―1r .@ •- 1@@r .@ - •
•@.-@ s-.-,- •, .@‘4@ •W@ . .@ L @•‘.@‘@ .:..@@ ::;@:-@@@ @t'@;@‘;@::@•@‘@k@@@ @-.‘-@ @,@ ,‘#@ 4 p.- FP:@f@ :@ :
@ 3@@@ I !
@ J:@ia,,YI@rti@@i14@:r@d@AIAi@ @S.e‘‘@@@
@@ •@@*4%@@@@ .@@ •@@‘
@@ .3 @r@q*I@+4*@@@,‘p,@I@p,# @#@/@ :4@@@ p;.. IS/d
@ t@r@@@@ I@@@ @.:@Vh
j;@@@ 4:,t' *.@@@ 4 @,@@ ‘@ •l,JI@@. •--,j•@@: @, - :.•r.: •• • 4@ : @‘@ 4?@@L@― •
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
FiG. 6.—Section through the cytoplasm of a HeLa cellgrown in 300 pg terramycin/ml for 24 hours. Numerous mitochondria have assumed a multilaminar appearance (1). Another mitochondrion is composed of compartmentalized vacuoles and encloses a dense myelin-like figure (2). The vacuolardistension of one mitochondrion has compressed the cristae toone side (3). X28,000.
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
-
@ •@,-
@,@1%@@
@.
4.
‘I
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
FIG. 7.—Low-power micrograph of HeLa cell from a culture exposed to 300 pg terramycin/ml for 24 hours. The cytoplasmic contents are condensed and consist of many vacuolatedand granular structures, derived from degenerate mitochondriaand vesiculated endoplasmic reticulum, enmeshed in fibrousmaterial. There are, as yet, no discernible changes in the structure of the nucleoli and nucleoplasm (nyc). X 10,000.
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
(@ I,,. -@,•
5).
@ -@ . I@
;S,'@ ,@;•‘@?@ :@. -, •@ ;@: -.
@•_@ .•@ •
@@ . •@- @\_•[email protected]•@::•‘,@•@-@•p'@ m@@@ t,@, •
@;[email protected]@••.@ ... .. .@ -
@ -:-@ . •.• -. j @-@. -..-t4@@@@ , t-(@
@, 4;.. 4--'.--. ‘:@ -@:-J@.' @t!'
L‘4 .(
‘s';@ I, â€-ç•-‘-d .@_@;•_ •‘,.4'-â€â€˜;,_-,‘..,I-. 4'@
‘ill
‘I 0
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
1963;23:551-554. Cancer Res L. J. Journey and Milton N. Goldstein MitochondriaThe Effect of Terramycin on the Fine Structure of HeLa Cell
Updated version
http://cancerres.aacrjournals.org/content/23/4_Part_1/551
Access the most recent version of this article at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://cancerres.aacrjournals.org/content/23/4_Part_1/551To request permission to re-use all or part of this article, use this link
on March 19, 2019. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from
