PROTON ATPase ARE INVOLVED IN METASTASIS IN HUMAN BREAST CANCER CELL by DEFENG LUG, M.S.. B.M.S. A THESIS IN PHYSIOLOGY Submitted to the Graduate Faculty of Texas Tech University Health Sciences Center in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Advisory Committee Raul Martinez-Zaguilan (Chairperson) Szindor Gyorke Narine Sarvazyan Accepted •v-..y .r* ^-^— '- — •-'- »» ^ »• — -»•.. —— Associate Dean of the Graduate School of Biomedical Sciences Texas Tech University Health Sciences Center December, 2001
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
PROTON ATPase ARE INVOLVED IN METASTASIS
IN HUMAN BREAST CANCER CELL
by
DEFENG LUG, M.S.. B.M.S.
A THESIS
IN
PHYSIOLOGY
Submitted to the Graduate Faculty of Texas Tech University Health Sciences Center
in Partial Fulfillment of the Requirements for the Degree of
for sample perfusion. The sample temperature was maintained at 37EC by keeping both
the water jacket and perfusion media at 37EC using an iso-temperature immersion
circulator water bath (Lauda model RM20, Brinkmann Instruments, Westbury, NY). All
measurements were performed using 4 nm-bandpass slits and an external rhodamine
standard as a reference. Fluorescence was monitored in continuous acquisition mode by
using an excitation wavelength of 534 nm and monitoring emissions at 584, 600, 644 nm
as described elsewhere (Martinez-Zaguilan et al, 1991). The fluorescence emission at
584 nm decreases and that at 644 nm increases with increasing pH; as such the ratio of
644/584 was used to monitor pH changes. The 600 nm wavelength, which is insensitive
to pH, was used to evaluate the efficiency of dye loading, quenching or other artifacts.
Fluorescence data were converted to ASCII format for analyses.
Spectral Ima^ng Microscope
Figure 2 shows the schematic representation ofthe major components required for
Spectral Imaging Microscopy (SIM). Briefly, an OLYMPUS IX 70 inverted microscope
was equipped with a 100 Watt Hg lamp as an illumination source. Imaging optics
20
included a 60X 1.4 NA OLYMPUS objective and a 5.4X eyepiece to focus the cell image
onto the input slit of a grating monochromator (Aries 250IS/SM spectrograph, Chromex,
Inc., Albuquerque, NM). A grating blazed at 300 lines/inch provided a spectral
bandwidth of 200 nm. The spectral output from the grating was imaged onto a liquid
cooled CCD camera (Photometries Mod. CH350, Tucson, AZ) equipped with a 512 x 512
element imaging chip that is 60% quantum efficient at 546 nm (Techtronics, Tucson,
AZ). The output image was composed of spectra acquired at multiple positions along the
length ofthe entrance slit. The spectral image occupied less than VA ofthe serial register
ofthe imaging chip and for a single cell image it occupies significantly less area.
Therefore, internal shifting of data allowed for rapid sequential image acquisition.
Adequate signal could be obtained with as little as 2 msec exposure time. Read out ofthe
full chip required 500 msec. Spectral resolution was primarily dependent on the grating
resolution, but also on the width ofthe entrance slit. As slit width was increased, signal
increases substantially while spectral resolution suffers comparably less (Martinez-
Zaguildn et al. 1996). Thus, spectral resolution was sacrificed for improved signal-to-
noise ratio. In the current experiments the entrance slit was set at 200 fim. Mapping ofthe
wavelength to position on the CCD chip was performed by reflecting light from the Hg
lamp to the monochromator. The grating was scanned to position the 610 line ofthe Hg
lamp near one end ofthe output spectrum and the doublet peak at 584/644 imaged near
the center. The digital output ofthe CCD camera was stored in a PC with 128 MB RAM.
Image analysis was done on a PC 300 MHz /128MB RAM using MAPS analysis
21
Eyepiece Spectrograph
Figure 2. Schematic illustration ofthe spectral imaging microscope system. The light source is alOO Watt mercury lamp(Hg Lamp). The specific excitation ofthe fluorophore is selected using anexcitation filter (EX. Filter). The emissions passes through an
adjustable entrance slit and be collected by the spectrograph with an attached Charge Coupled Device (CCD) camera, which connected to a computer. Using software MAPS-2 the computer give out the spectra ofthe emissions captured.
22
software version 2.0 (Photometries, Tucson, AZ) and further analyzed using MS Excel
2000 (Microsoft Co., Redmond, WA) and Sigma Plot for Windows, version 5.0 (Jandel
Scientific, San Rafael, CA) (Figure 3).
Subconfluent cultures of breast cancer cells were grown on 25 mm round glass
coverslips loaded with 7 fiM SNARF-l/AM as mentioned in fluorescence spectroscopy
experiment. Then the coverslips were mounted onto a special chamber and placed in a
special temperature controlled perfiasion stage PDMI-2 (Medical Systems Inc.,
Greenvale, NY), with a buffer perfusion rate of 3 ml/min. This was placed on the sample
stage of 1X70 Olympus inverted microscope.
Laser Scanning Confocal Microscope
Confocal Microscopy was performed with a Bio-Rad MRC1024 confocal
microscope (Rojas et al. 2000). Subconfluent breast cancer cells were loaded with 7 |iM
SNARF-l/AM and mounted to the same chamber and temperature controlled perfusion
stage as described above for spectral imaging microscope. A single cell was visualized in
each coverslip. Line scans were carried out by drawing a line across the cell from the
leading edge to the lagging edge. Four regions of interest were selected on the line for
each cell. Using 514 nm argon laser for excitation, the emissions were collected at 584
nm and 644nm from the interest regions every 2 milli-seconds during scanning (Figure
4). The data was acquired on an 0S2 based computer using tiie Lasersharp software.
Intracellular pHs were calculated with the ratio of two emissions (644nm/584nm) and
pKa, Rmin and R ax from the in situ titration in the same regions ofthe cells in the fiirther
23
o
^^BSH^-.»ju_x—/^^— A o o x ' ^ ^ ^ ^ ^ ^ B • 1 d.U.T. 1 ex—4oo I ^ H ft'O O O o -^^1 f-~^ ° ° <=> ^ H ELPS n m ^ \ ^ H
• V ft.^.i. ' ' ' ' s ^ J K T j^jf^ -Bff" -^^^"ji^^
W ^.jf" T, -5
H " B T I
!•_. ^^JjV' H K ^ ^ ' ^ S..
|H|^ ^ " 7 ^a;
5 ; . m
o ' ^T l
• 5 1
CD ^
CO . ^ J l
» « 1 2d • o - - -Ji —ii> -, — i . |
— — 1 ^ ^^ f7 '^^^1 o o o l ^ H <l> ^ CM ~ ^ ^ l CO «0 CO Jj
|-TA88*«^)r4^^^i^e--^^
0) o c 03
LU
(88t^=X3)'i'n'B
^ c
:^ is
2 ex < .
Ji § s a. o w a,
o OQ
s ••-»
c: o _ (U (U
a, o <0 trt
— CO
to C
J-i (/3
o U)
w <u o
c .S
CD
CO o ^
tz; w TS
24
< z
(D
X
• I -H
o
o • » ^ - * — >
a
3
in 4—>
C
-5
a
X
o
P3 cd
>
u
O
c
C<3 O
c o U c o o c ".J
UH
3 5£l
25
data analysis with MS Origin® statistical software and Sigma Plot for Windows,
5.0 (Jandel Scientific, San Rafael, CA) (detail in data analysis).
version
Immunocytochemistry
Monoclonal antibodies to several subunits of V-H^ATPase are available from
Figure 6. Bafilomycin A,(Baf) inhibits invasion ofthe breast cancer cells Cells were loaded with Calcein and plated in HTS Fluoro-Block inserts for 48 hours in the presence or abscence of 50nM bafilomycin A,. The uiserts were visualized under LSCM. Stars show significant difference between no Baf and with Baf (n =5, p<0.05).
36
NH.CI Na"- Free
8.0 .
MB231
MB435S MCF-7 ^
5 min
MCF-7
MB435S
IVB231
0.
B
1-H
-
0 0.3 0.6 0.9
J^ (nrMmin)
Figure 7. Highly Invasive Cells Exhibit Larger Proton Fluxes (JH+) Than Lewdly Invasive Cells. A. Acid loading experiments in cell populations. Cells v^ere loaded with SNARF-1. Tthe ratios of both emission 644/584 were measured. Intracellular pHs were calculated. B. Proton fluxes were quantified as mentioned in chapter II. Highly invasive breast cancer cells
have larger proton flux than lowly invasive(n=ll , differences are significant between groups, p<0.05). C. Bafilomycin Al inhibits proton flux in MB231 cells data are representative of 7 experiments..
37
In order to further confirm that proton fluxes were produced by V-H""ATPase,
bafilomycin Ai was introduced into the Na free and Ca free CSB. Data showed that
bafilomycin Ai reduced the proton fluxes in MDA-MB-231 cells (Figure 7c).
Combining these results with the data from the invasion and migration assays (MDA-
MB-23 1 cell is more invasive than MCF-7 cells), we conclude that highly invasive breast
cancer cells have more proton pump activities. These results also supported indirectly
that V-H^ ATPase maybe involved in the invasion ofthe cancer cells.
Intracellular pH Gradients Are Larger in Highlv Invasive
Breast Cancer Cells
It is well known that cancer cells live in an acidic environment and the pHin is
alkaline. We also know from the present study that V-H"^ATPases are expressed on the
plasma membrane ofthe breast cancer cells. The immunocytochemistry data indicated
that the distribution of this enzyme is not uniform. Based on this, we hypothesize that the
pHin of breast cancer cells is not evenly distributed either. To unveil this secret, line
scanning confocal microscopy (LSCM) was employed to measure the pH.n- In the present
study, we drew a line from the leading edge to the lagging edge ofthe cells. Emissions of
SNARF-1 were collected from four points on the line (Figure 3) every 2 milli-seconds.
Intracellular pH values were calculated as mentioned in data analysis. Chapter II. Data
showed that pHjn is not equally distributed in the breast cancer cells; i.e., pHin gradients
did exist. The pHin at the leading edge ofthe cell was more alkaline than that at the
lagging edge. Intracellular pH gradients were larger in the highly invasive cancer cells
38
Hd
C/D
"Q3 o (D > ^
*3 m 5 rsi c S9 •^ S > . ' * ^ ^ ^ ^ ^ ^ ^ I M I ^ ^
- ^ r^w bX) / ^ • ^ i ^ ^ X
K ^ o ^
edge
in
g
OG t>
cd 1
J 1
edge
-—0
Lea
di
•
-
•
I
a5 o6 Hd
j^ c ^
'-> -J '-> tL
? 3 ' J C ^ r3 Z^ ' J 'jz:.
y. -^ — O
x; _c: ) •
•4—«
i i C C^
« ^ v • " •
s ^ -£ 5 C/5 " =
C/3
> ^ ' ! A O r3 <-, > — r - • " "
• ~ C3
>^T3
^ i Q
r- S r3 '^
-^ ^ ;|; u
• •6 ^ ' ^ 1
u* - ^
^ B — CJ
^ iJD
~ <— a .~ t C V2
Z^" :.s < x: ;!:: o , -y; U ,
= csi
8< o Z > c/
> :2
c r->%-5
• ^ • ^ M
SD ^
— CJ
. -a 3C ^
CJ ^
= . _sp o
CJ
c c CJ
/^ vL •~j
<—
.^^ 'J
,o • • * •
r -
c C3 o v: CJ r-
1 ^
" 3
CJ
CJ
CJ
^ CJ
_3P CJ
tXj
• K M
_C3
-a ra SD
• — • a C3
CJ r "
^ s ^
y: r;
C/5
"s (U (U
J= h-Cj" SD
CJ
SD c
"sb SD W>J
CJ
•^ c y"^
"a CJ
^^
j j
V V
J = CJ
39
(Figure 8a), such as MD-MB-231 cells, than in the lowly invasive cancer cells, such as
MCF-7 (Figure 8b). Proton fluxes were larger in the leading edge than in the lagging
edge. This implied that more proton pump activities occurred in the leading edge ofthe
cancer cells and it was more prominent in the highly invasive cell line.
Proton Pump Activities Are Elevated in the Leading Edge ofthe Breast Cancer Cells
Spectral imaging microscope (SIM) can also measure the pHin ofthe single cell.
In this experiment, breast cancer cells were loaded with SNARF-1, single cell was chosen
under 60x oil objective the cell was orientated from the leading edge to the lagging edge.
First order images were obtained with a 610 nm emission filter. The entrance slit ofthe
spectrograph (Figure 2) was narrow down from 2000 pm to 200pm and the emissions
were collected by reading sixteen spectra from the slit. The pH measurements of 16
intracellular areas in the slit were obtained by the data analysis afterwards. The pH
gradients were consistent with our observation from the LSCM. The leading edge ofthe
cancer cell had a more alkaline pH than the lagging edge. In the recovery stage ofthe
NH4CI acid loading experiment, proton flux was larger in the leading edge than in the
lagging edge (Figure 9). This fiirther confirmed the results ofthe LSCM.
Bafilomycin Sensitive ATPase Activities Were Discovered in the Plasma Membrane Fractions ofthe Breast Cancer Cells
V-H^ATPases have been found on the cell membrane immunocytochemically and
physiologically in all the experiments in the present study. To more directly address this
40
issue, we then performed biochemical studies. Breast cancer cells were cultured to
confluency and harvested. After homogenization, the membrane fraction ofthe cells was
used for the ATPase assay. The ATPase activities measured from the membrane fraction
ofthe breast cancer cells without drug treatment served as the control (100%). In the
experimental groups, membrane fractions were preincubated with 50 nM bafilomycin Ai.
or with both 50 nM bafilomycin Ai and 5 pM ouabain. Results show that bafilomycin Ai
significanfly decreased the ATPase activity MDA-MB-468 and MDA-MB-231 cells as
compared with the control groups. Bafilomycin Ai sensitive ATPase activities composed
25 - 45% ofthe total ATPase activities on the cell membranes ofthe breast cancer cells.
Of membrane fractions preincubated with both bafilomycin Ai and ouabain (5 pM), the
ATPase activities tended to be even lower (Figure 10), which suggested that the
bafilomycin sensitive ATPase activity was something other than Na^, K"'-ATPase.
41
7.5
7.0
a 6.5-
6.0-
0 5 10 15 20 25 30 35 Time (min)
Figure 9. Spectral imaging microscope show intracellular pH gradients exist from leading edge to lagging edge ofthe MDA-MB231 breast cancer cells. The leading edge ofthe cell have a more alkaline pH.
42
CO CN
I
cn
_C/)
o 00 CD - ^
I
GO
CJ
CJ
CJ
CJ y)
QJ
>
C/3
c
<
CQ
y5 II
C/3 —
•^ (U
OJ o
Cu CJ
c OS CJ
5 00 (O CM
(o/o) X;iAipv ssBdlv
43
^^^m^^
CHAPTER IV
DISCUSSION AND CONCLUSION
V-H ATPase and Tumor Invasive Behavior
Tumor invasion and metastasis are two hallmarks ofthe neoplasm malignancy.
They are the major causes ofthe morbidity ofthe cancer patients. Liotta et al. (1986)
delineated events related to the process of invasion ofthe extracellular matrix, including
the basement membrane, by tumor cells. These events include: (a) attachment of tumor
cells to laminin or fibronectin via cell surface receptors for these molecules; (b) local
degradation ofthe matrix by tumor cell-associated proteases; and (c) tumor cell
locomotion through the matrix, assisted by proteolysis. The invasive capacity ofthe
tumor cell can be examined in vitro by amnion invasion assay (Yagel et al. 1989) and
Matrigel assay (Hendrix et al. 1987). Many factors maybe involved in this process, such
as Ca2 (Marks et al. 1990, Milne et al. 1991, Savarese et al. 1992, Korczak et al. 1989),
chemoattractants (Miller et al. 1990, Mareel et al. 1990), and several proteases, e.g.
collagenase (Yagel et al. 1989, Stetler-Stevenson 1990), cathepsins (Lah et al. 1992,
Rozhin et al. 1994), metalloproteinases (Matrisian et al. 1992) and serine protease (Fidler
1991, Vassalli et al. 1991), etc. No direct evidence for V-H" ATPase involvement in
tumor invasion has been documented yet. However, the relation between proton
concentration (pH) and invasion has drawn extensive attention. Firstly, all the proteases
mentioned above are pH sensitive. Cathepsins are lysosomal enzymes, which prefer an
acidic pH (Morisset et al. 1986). Acidic pH induces the redistribution and release of
44
cathepsin B from a series of metastatic human cell lines (Rozhin et al. 1994).
Mathematical models have been used to investigate whether altered proteolytic activity at
low pH is responsible for the stimulation of a more metastatic phenotype. Webb et al.
(1999) examined the effect of culture pH on the secrefion and activity of two different
classes of proteinases: the metalloproteinases (MMPs) and the cysteine proteinases (such
as cathepsin B). The mathematical modeling suggested that changes in MMP activity at
low pH did not significantly affect invasive behavior. However, the model predicted that
the levels of active cathepsin B were significantly altered by acidic pH. This result
suggested a critical role for the cysteine proteinases in tumor progression (Webb et al.
1999). Acidic pH directly associated with invasive behavior of tumors was noticed
recently by Martinez-Zaguilan et al. (1996). In this research, the in vitro invasive
potential of two human melanoma cell lines, the highly invasive C8161 and poorly
invasive A375, were examined. Culturing of either cell line at acidic pH (6.8) caused
dramatic increases in both migration and invasion, as measured with the Membrane
Invasion Culture System (MICS). These data indicated that culturing of cells at mildly
7.+
acidic pH induced them to become more invasive. Intracellular calcium ([Ca ];„) was
also examined in this experiment but it was not consistent with invasive potenfial
(Martinez-Zaguilan et al. 1996). Tumor cells have a lower extracellular pH (pHcx) than
normal cells; this is an intrinsic feature ofthe tumor phenotype, caused by alterafions
either in acid exports from the tumor cells or in clearance of extracellular acid. Low pHex
benefits tumor cells because it promotes invasiveness, whereas a high pHin gives them a
competitive advantage over normal cells for growth. Molecular genetic approaches have
45
revealed hypoxia-induced coordinated upregulation of glycolysis, a potentially important
mechanism for establishing the metabolic phenotype of tumors (Stubbs et al. 2000). A
combined result of mathematical analyses, experimental data, and clinical observations
also supported the hypothesis that tumor-induced alteration of microenvironmental pH
may provide a simple but complete mechanism for cancer invasion (Gatenby et al. 1996).
Controversies also exist in this field. Cuvier et al. (1997) examined whether the
invasive capacity ofthe cells could be influenced by hypoxia, glucose starvation and
acidosis with a Matrigel, a basement membrane-like preparation, in a two-chamber
invasion assay to address this issue. Both KHT-LPl, a murine sarcoma cell line, and
SCC-VII, a murine squamous carcinoma cell line, showed an increased ability to invade
through Matrigel after hypoxia and glucose starvation, but there was no consistent change
in invasive capacity following acidosis exposure. They also found that cathepsin (L+B)
content varied according to the cell line and the treatment received (hypoxia, glucose
starvation). There was an increase of cathepsin content for KHT-LPl cells exposed to
hypoxia which correlated well with the increase ofthe invasion ability through Matrigel.
No increase of cathepsin content was observed for hypoxia-treated SCC-VII or for KHT-
LPl and SCC-VII cells treated with glucose starvation. These results suggested that
transient hypoxia and glucose starvation, but not acidosis, can increase the invasive
ability of tumor cell lines.
Evidence of V-H^ATPase involved in microvascular invasion was found recently
in our lab. Microvascular endothelial cells thrive in an acidic environment, suggesting
that they must exhibit a dynamic pH^ regulatory mechanism to cope with acidosis. The
46
results showed that these cells: (a) exhibited pmV-H""ATPase activity; (b) expressed
pmV-H"*"ATPase activity at the leading edge during migration; (c) exhibited a more
alkaline pHin at the leading edge of migrating cells; and (d) migrafion and invasion of
microvascular endothelial cells were inhibited by bafilomycin Ai. Altogether, these data
suggested that pmV-H" ATPases were essential for invasion and migration, the two
essential steps in angiogenesis (Rojas et al. 2001). V-H" ATPases proved important for
acidification-dependent degradation of tissue matrices, through which some cell types
move, and for pH regulation across some epithelial cell layers. Placentation involved
intricate signaling, cell proliferation, and controlled invasion. Redistribution V-
H^ATPase was observed and found to be stage-dependent during the bovine
implantation. Epithelial expression of all three subunits of V-H"* ATPase was observed,
and in nonpregnant animals this expression was apical. As pregnancy proceeded,
expression of all subunits became pericellular in luminal but not glandular epithelium.
The trophoblast expressed all three subunits during initial contact with the epithelium. In
the stroma, ductin (the 16kD subunit of V-H^ATPase) expression was reduced after
implantation. This suggested that ductin plays a role in the shifting communication
between stromal and epithelial cells induced by embryo attachment (Skinner et al. 1999).
In Kubota et al.'s study (2000), they established transfectants over-expressing the V-
H^ATPase 16 kDa subunit at the mRNA level, and found that these transfectants showed
an enhanced invasiveness through Matrigel with concomitant increases in secretion of
with a maximum stoichiometry of 1 mol of V-H"* ATPase per 8 mol of F-actin and an
apparent affinity of 0.05 pM. Electron microscopy of negatively stained samples
confirmed the binding interaction (Powell et al. 2000). These findings linked transport of
V-H^ATPase to reorganization ofthe actin cytoskeleton during osteoclast activation.
Actin and myosin are two important components of cell movement (Albert et al. 1994).
Montcourrier et al.(1994) examined the large acidic vesicles of human metastatic
breast cancer cells in culture by transmission electron microscopy, which measured pH of
50
the vesicles by video-enhanced epifluorescence using FITC-dextran. They found that the
presence ofthe large vesicles in metastatic MDA-MB-231 cells correlated with an
increased ability of cells to migrate through Matrigel and a high cathepsin D
concentration. These cells were able to phagocytize 1.24-micron latex beads and
fluorescent Matrigel and incorporate this extracellular material into large acidic vesicles.
Large acidic vesicles were actively acidified with an H^-ATPase vacuolar pump
specifically inhibited by bafilomycin A]. This indicated that large acidic vesicles were
associated with both phagocytosis and invasion. Montcourrier et al concluded that the
phagocytotic activity of breast cancer cells, associated with high cathepsin D expression
and high acidification potential, characterized cancer cells that migrated through
Matrigel.
In conclusion, the present study showed that more V-H^ATPases are expressed at
the surface ofthe highly invasive breast cancer cells than in the lowly invasive cells.
Invasion of metastatic cells was inhibited by bafilomycin Ai a V-H+ATPase inhibitor.
The J ^^ was more elevated in highly invasive cells than in lowly invasive breast cancer
cells. Breast cancer cells exhibited a more alkaline pH in the leading edge than in the
lagging edge. Intracellular pH gradients were larger in the highly than in the lowly
invasive cells. A complete understanding ofthe mechanism by which V-H"" ATPase
works in invasion and metastasis require ftirther study. However, energization ofthe
movement of molecules across the cell membrane and changing the pHi„ consequenfly
activates the proteins related to cell motility maybe one ofthe possible mechanism.
51
REFERENCES
Abrahams JP, Leslie AG, Lutter R, Walker JE. Structure at 2.8 A resolution of Fr ATPase from bovine heart mitochondria [see comments]. Nature 1994; 370:621-628.
Adachi I, Puopolo K, Marquez-Sterling N, Arai H, Forgac M. Dissociation, cross-linking, and glycosylation ofthe coated vesicle proton pump. J Biol Chem 1990; 265:967-973.
Albert B, Bray D, Lewis J Raff M, Robert K, Watson JD. (1994) The cytoskeleton in Molecular Biology ofthe Cell, 3"* ed. pp 788-858
Altenberg GA, Young G, Horton JK, Glass D, Belli JA, Reuss L. Changes in intra- or extracellular pH do not mediate P-glycoprotein-dependent multidrug resistance. Proc Nad Acad Sci U.S.A. 1993; 90:9735-9738.
Andersson GN, Tomdal UB, Eriksson LC. Decreased vacuolar acidification capacity in drug-resistant rat liver preneoplastic nodules. Cancer Res 1989; 49:3765-3769.
Anraku Y, Umcmoto N, Hirata R, Wada Y. Structure and function ofthe yeast vacuolar membrane proton Al Pase. J Bioenerg.Biomembr. 1989; 21:589-603.
Blair HC, Teitelbaum SL, Ghiselli R, Gluck S. Osteoclastic bone resorption by a polarized vacuolar proton pump. Science 1989; 245:855-857.
Boekema EJ, Ubbink-Kok T, Lolkema JS, Brisson A, Konings WN. Visualization of a peripheral stalk in V-type ATPase: evidence for the stator structure essential to rotafional catalysis. Proc Natl Acad Sci U.S.A. 1997; 94:14291-14293.
Boscoboinik D, Gupta RS, Epand RM. hivestigation ofthe relationship between altered intracellular pH and multidrug resistance in mammalian cells. Br.J Cancer 1990; 61:568-572.
Bowman EJ, Tenney K, Bowman BJ. Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-1 encoding the 67-kDa subunit reveals homology to other ATPases. J Biol Chem 1988; 263:13994-14001.
Bowman EJ, Siebers A, Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Nad Acad Sci U.S.A. 1988;85:7972-7976.
52
Brown D, Lui B, Gluck S, Sabolic I. A plasma membrane proton ATPase in specialized cells of rat epididymis. Am J Physiol 1992; 263:C913-C916
Cassel D, Katz M, Rotman M. Depletion of cellular ATP inhibits Na+/H+ antiport in cultured human cells. Modulation ofthe regulatory effect of intracellular protons on the antiporter activity. J Biol Chem 1986; 261:5460-5466.
Chatterjee D, Chakraborty M, Leit M, et al. Sensitivity to vanadate and isoforms of subunits A and B distinguish the osteoclast proton pump from other vacuolar H+ ATPases. Proc Nad Acad Sci U.S.A. 1992; 89:6257-6261.
Chu S, Brownell WE, Montrose MH. Quanritative confocal imaging along the crypt-to-surface axis of colonic crypts. Am. J. Physiol. 1995; 269:C1557-C1564
Clague MJ, Urbe S, Aniento F, Gruenberg J. Vacuolar ATPase activity is required for endosomal carrier vesicle formafion. J Biol Chem 1994; 269:21-24.
Cross RL, Taiz L. Gene duplication as a means for altering H+/ATP ratios during the evolution of Fo Fi ATPases and synthases. FEBS Lett 1990; 259:227-229.
Cross RL, Duncan TM. Subunit rotation in FQFI-ATP synthases as a means of coupling proton transport through Fo to the binding changes in Fi. J Bioenerg. Biomembr. 1996;28:403-408.
Cuvier C, Jang A, Hill RP. Exposure to hypoxia, glucose starvation and acidosis: effect on invasive capacity of murine tumor cells and correlation with cathepsin (L + B) secretion. Clin Exp Metastasis 1997; 15:19-25.
Fidler IJ. Cancer metastasis. Br. Med Bull. 1991; 47:157-177.
Fillingame RH. Coupling H+ transport and ATP synthesis in Fi FQ-ATP synthases: glimpses of interacting parts in a dynamic molecular machine. J Exp Biol 1997; 200:217-224.
Forgac M. Structure, ftinction and regulafion ofthe vacuolar (H+)-ATPases. FEBS Lett 1998;440:258-263.
Forgac M. Structure and properties ofthe vacuolar (H+)-ATPases. J Biol Chem 1999; 274:12951-12954.
Gatenby RA, Gawlinski ET. A reaction-difftision model of cancer invasion. Cancer Res 1996; 56:5745-5753.
Gillies RJ, Ogino T, Shulman RG, Ward DC. 3 IP nuclear magnetic resonance evidence for the regulation of intracellular pH by Ehrlich ascites tumor cells. J Cell Biol 1982;95:24-28.
53
Gillies RJ, Martinez-Zaguilan R, Martinez GM, Serrano R, Perona R. Tumorigenic 3T3 cells maintain an alkaline intracellular pH under physiological conditions Proc NaU Acad Sci U.S.A. 1990; 87:7414-7418.
Gillies RJ, Martinez-Zaguilan R. Regulation of intracellular pH in BALB/c 3T3 cells. Bicarbonate raises pH via NaHC03/HCl exchange and attenuates the activation of Na+/H+ exchange by serum. J Biol Chem 1991; 266:1551-1556.
Gluck SL. The sttaicUire and biochemistry ofthe vacuolar H+ ATPase in proximal and distal urinary acidification. J Bioenerg.Biomembr. 1992;24:351-359.
Goldstein DJ, Finbow ME, Andresson T, et al. Bovine papillomavirus E5 oncoprotein binds to the 16K component of vacuolar H(+)-ATPases. Nature 1991; 352:347-349.
Gottesman MM, Pastan I. Biochemistry of multidrug resistance mediated by the muUidrug transporter. Annu Rev Biochem 1993; 62:385-427:385-427.
Gottlieb RA, Giesing HA, Zhu JY, Engler RL, Babior BM. Cell acidification in apoptosis: granulocyte colony-stimulating factor delays programmed cell death in neutrophils by up-regulating the vacuolar H' -ATPase. Proc Nad Acad Sci U.S.A. 1995;92:5965-5968.
Griffiths JR. Are cancer cells acidic? [editorial]. Br.J Cancer 1991; 64:425-427.
Gunn JM, Martinez-Zaguildn R, Wald-Hopkins S, Woolridge D, Gillies RJ. NIH3T3cells transfected with the yeast H(+)-ATPase have altered rates of protein tumover. Arch Biochem Biophys 1994; 314:268-275.
Harvey WR, Wieczorek H. Animal plasma membrane energization by chemiosmotic H+ V-ATPases. J Exp Biol 1997; 200:203-216.
Heinz A, Sachs G, Schafer JA. Evidence for activation of an active electrogenic proton pump in Ehrlich ascites tumor cells during glycolysis. J Membr. Biol 1981; 61:143-153.
Hendrix MJ, Seftor EA, Seftor RE, Fidler IJ. A simple quantitative assay for studying the invasive potential of high and low human metastatic variants. Cancer Lett 1987; 38:137-147.
Hirata R, Graham LA, Takatsuki A, Stevens TH, Anraku Y. VMAl 1 and VMA16 encode second and third proteolipid subunits ofthe Saccharomyces cerevisiae vacuolar membrane H- -ATPase. J Biol Chem 1997; 272:4795-4803.
54
Hwang ES, Nottoli T, Dimaio D. The HPV16 E5 protein: expression, detection, and stable complex formation with transmembrane proteins in COS cells Virology 1995;211:227-233.
Juranka PF, Zastawny RL, Ling V. P-glycoprotein: multidrug-resistance and a superfamily of membrane-associated transport proteins. FASEB J 1989- 3-2583-2592.
Kane PM. Regulation of V-ATPases by reversible disassembly. FEBS Lett 2000.Mar.l0.;469.(2.-3.):137.-41. 469:137-141.
Kane PM. Disassembly and reassembly ofthe yeast vacuolar H(+)-ATPase in vivo. J Biol Chem 1995; 270:17025-17032.
Kane PM. Introduction: V-ATPases 1992-1998. J Bioenerg.Biomembr. 1999;31:3-5.
Keizer HG, Joenje H. Increased cytosolic pH in multidrug-resistant human lung tumor cells: effect of verapamil. J Nati Cancer Inst. 1989; 81:706-709.
Korczak B, Whale C, Kerbel RS. Possible involvement of Ca2+ mobilization and protein kinase C activation in the induction of spontaneous metastasis by mouse mammary adenocarcinoma cells. Cancer Res 1989; 49:2597-2602.
Kraus M, Severin T, Wolf B. Relevance of microenvironmental pH for self-organized tumor growth and invasion. Anticancer Res 1994; 14:1573-1583.
Kubota S, Seyama Y. Overexpression of vacuolar ATPase 16-kDa subunit in lOTl/2 fibroblasts enhances invasion with concomitant induction of matrix metalloproteinase-2 [In Process Citation]. Biochem Biophys Res Commun 2000.NOV. 1900;278:390-394.
Lah TT, Kokalj-Kunovar M, Strukelj B, et al. Stefins and lysosomal cathepsins B, L and D in human breast carcinoma. Int J Cancer 1992; 50:36-44.
Lee BS, Gluck SL, Holliday LS. Interaction between vacuolar H (+)-ATPase and microfilaments during osteoclast activation. J Biol Chem 1999; 274:29164-29171.
Liotta LA, Rao CN, Wewer UM. Biochemical interactions of tumor cells with the basement membrane. Armu Rev Biochem 1986; 55:1037-57:1037-1057.
Liu Q, Kane PM, Newman PR, Forgac M. Site-directed mutagenesis ofthe yeast V-ATPase B subunit (Vma2p). J Biol Chem 1996; 271:2018-2022.
Lowry OH, Rosebrough NJ, Farr AL and Randall RJ. J. Biol. Chem 1951; 193: 265-275.
55
Lynch RM, Camngton W, Fogarty KE, Fay FS. Metabolic modulation of hexokinase association with mitochondria in living smooth muscle cells. Am J Physiol 1996;270:C488-C499
Manabe T, Yoshimori T, Henomatsu N, Tashiro Y. Inhibitors of vacuolar-type H (+)-ATPase suppresses proliferation of culUired cells. J Cell Physiol 1993; 157:445-452.
Mareel MM, Van Roy FM, De Baetselier P. The invasive phenotypes. Cancer Metastasis Rev 1990;9:45-62.
Marks PW, Maxfield FR. Transient increases in cytosolic free calcium appear to be required for the migration of adherent human neutrophils [published erratum appears in J Cell Biol 1990 Mar; 110(3): 861]. J Cell Biol 1990; 110:43-52.
Marquardt D, Center MS. Involvement of vacuolar H (+)-adenosine triphosphatase activity in multidrug resistance in HL60 cells. J NaU Cancer Inst. 1991; 83:1098-1102.
Marsh W, Sicheri D, Center MS. Isolation and characterization of adriamycin-resistant HL-60 cells which are not defective in the initial intracellular accumulation of drug. Cancer Res 1986;46:4053-4057.
Martinez-Zaguilan R, Martinez GM, Lattanzio F, Gillies RJ. Simultaneous measurement of intracellular pH and Ca2-i- using the fluorescence of SNARF-1 and ftira-2. Am. J. Physiol. 1991; 260:C297-C307
Martinez-Zaguildn R, Lynch RM, Martinez GM, Gillies RJ. Vacuolar-type H (+)-ATPases are functionally expressed in plasma membranes of human tumor cells. Am J Physiol 1993; 265:C1015-C1029
Martinez-Zaguildn R, Seftor EA, Seftor RE, Chu YW, Gillies RJ, Hendrix MJ. Acidic pH enhances the invasive behavior of human melanoma cells. Clin Exp Metastasis 1996; 14:176-186.
Martinez-Zaguilan R, Martinez GM, Gomez A, Hendrix MJ, Gillies RJ. Distinct regulation of pHin and [Ca2+]in in human melanoma cells with different metastatic potential. J Cell Physiol 1998; 176:196-205.
Martinez-Zaguilan R, Raghunand N, Lynch RM, et al. pH and drug resistance. I. Functional expression of plasmalemmal V-type H+-ATPase in drug-resistant human breast carcinoma cell lines. Biochem Pharmacol 1999; 57:1037-1046.
56
Martinez GM, Martinez-Zaguilan R, Gillies RJ. Effect of glucose on pHin and [Ca2+]^ m NIH-3T3 cells transfected with the yeast P-type H (+)-ATPase. J Cell Physiol 1994; 161:129-141. ' v / /
Matnsian LM. The matrix-degrading metalloproteinases. Bioessays 1992; 14:455-463.
McGrath T, Center MS. Adriamycin resistance in HL60 cells in the absence of detectable P-glycoprotein. Biochem Biophys Res Commun 1987; 145:1171-1176.
Miller FR, Heppner GH. Cellular interactions in metastasis. Cancer Metastasis Rev 1990;9:21-34.
Mdne JL, Coukell MB. A Ca2+ transport system associated with the plasma membrane of Dictyostelium discoideum is activated by different chemoattractant receptors. J Cell Biol 1991; 112:103-110.
Montcourrier P, Mangeat PH, Valembois C, et al. Characterization of very acidic phagosomes in breast cancer cells and their association with invasion. J Cell Sci 1994; 107:2381-2391.
Morisset M, Capony F, Rochefort H. The 52-kDa estrogen-induced protein secreted by MCF7 cells is a lysosomal acidic protease. Biochem. Biophys. Res. Commun. 1986; 138:102-109.
Nelson N. Structural conservation and functional diversity of V-ATPases. J Bioenerg.Biomembr. 1992; 24:407-414.
Nelson N, Harvey WR. Vacuolar and plasma membrane proton-adenosinetriphosphatases. Physiol Rev 1999; 79:361-385.
Nishi T, Forgac M. Molecular cloning and expression of three isoforms ofthe 100-kDa a subunit ofthe mouse vacuolar proton-translocating ATPase. J Biol Chem 2000.Mar.lO; 275. (10.): 6824.-30. 275:6824-6830.
Nuccitelly R and Heiple JM. In Intracellular pH: Its Measurement Regulation and Utilization in Cellular fianction (eds Nuccitelly R and Deamer DW) pp 567-586 Liss New York, 1982)
Ober SS, Pardee AB. Intracellular pH is increased after transformation of Chinese hamster embryo fibroblasts. Proc Nati Acad Sci U.S.A. 1987; 84:2766-2770.
Parker SL, Tong T, Bolden S, Wingo PA. Cancer statistics, 1997. CA. Cancer J. Clin. 1997;47:5-27.
57
Perin MS, Fried VA, Stone DK, Xie XS, SudhofTC. Structtire ofthe 116-kDa polypeptide ofthe clathrin-coated vesicle/synaptic vesicle proton pump. J Biol Chem 1991;266:3877-3881.
Perona R, Serrano R. Increased pH and tumorigenicity of fibroblasts expressing a yeast proton pump. Nature 1988; 334:438-440.
Perona R, Portillo F, Giraldez F, Serrano R. Transformation and pH homeostasis of fibroblasts expressing yeast H (+)-ATPase containing site-directed mutations. Mol Cell Biol 1990; 10:4110-4115.
Peterson EP, Martinez GM, Martinez-Zaguildn R, Perona R, Gillies RJ. NIH 3T3 cells transfected with a yeast H (+)-ATPase have altered sensitivity to insulin, insulin growth factor-I, and platelet-derived growth factor-AA. J Cell Physiol 1994; 159:551-560.
Pressley TA, Haber RS, Loeb JN, Edelman IS, Ismail-Beigi F. Stimulation of Na, K-activatcd adenosine triphosphatase and active transport by low external K+ in a rat liver cell line. J. Gen. Physiol. 1986; 87:591-606.
Putnam RW. Intracellular pH Regulation (1997) in Cell Physiology Source Book 2"" edition (Sperclakis N ed), pp 293-311 Academic Press San Diego London Boston New York Sydney Tokyo Toronto
Raghunand N, Martinez-Zaguildn R, Wright SH, Gillies RJ. pH and drug resistance. II. Tumover of acidic vesicles and resistance to weakly basic chemotherapeutic drugs. Biochem Pharmacol 1999;57:1047-1058.
Ries LAG, Kosary CL, Hankey BF, Miller BA, Edwards BK, eds. SEER Cancer Statistics Review, 1973-1995. National Cancer Institute, Bethesda, MD, 1998
Rojas JD, Sanka SC, Martinez GM, Seftor EA, Meininger C J, Wu G, Wesson DE, Hendrix MJC, Martinez-Zaguildn R. Plasmalemmal Vacuolar Type H' -ATPases Mediate Migration/Invasion and Maintain pH Gradients from Leading to Lagging Edge in Microvascular Endothelial Cells. Cell (in print)?
Rojas JD, Sanka SC, Luo D, Bush C, Martinez GM, Hendrix MJC and Martinez-Zaguilan R. Proton pumps, Angiogenesis, and Metastatic Breast Cancer. Proc Soc Int Engin SPIE, 3924: 67; 78, 2000
58
Roninson IB. Molecular mechanism of multidrug resistance in ttimor cells. Clin Physiol Biochem 1987;5:140-151.
Roos A, Boron WF. Intracellular pH. Physiol. Rev. 1981; 61:296-434.
Rozhin J, Sameni M, Ziegler G, Sloane BF. Pericellular pH affects distribution and secretion of cathepsin B in malignant cells. Cancer Res 1994; 54:6517-6525.
Savarese DM, Russell JT, Fatatis A, Liotta LA. Type IV collagen stimulates an increase m intracellular calcium. Potential role in ttimor cell motility. J Biol Chem 1992; 267:21928-21935.
Schindler M, Grabski S, Hoff E, Simon SM. Defective pH regulation of acidic compartments in human breast cancer cells (MCF-7) is normalized in adriamycin-resistant cells (MCF-7adr). Biochemistry 1996; 35:2811-2817.
Stetler-Stevenson WG. Type IV collagenases in tumor invasion and metastasis. Cancer Metastasis Rev. 1990; 9:289-303.
Stevens TH, Forgac M. Structure, function and regulation ofthe vacuolar (H+)-ATPase. Annu Rev Cell Dev. Biol 1997; 13:779-808:779-808.
Stubbs M, McSheehy PM, Griffiths JR, Bashford CL. Causes and consequences of tumour acidity and implications for treatment. Mol Med Today 2000.Jan.; 6.(l.):15.-9. 6:15-19.
Stubbs M, Veech RL, Griffiths JR. Tumor metabolism: the lessons of magnetic resonance spectroscopy. Adv.Enzyme Regul. 1995; 35:101-15:101-115.
Swallow CJ, Grinstein S, Rotstein OD. A vacuolar type H(+)-ATPase regulates cytoplasmic pH in murine macrophages. J Biol Chem 1990; 265:7645-7654.
Swallow CJ, Grinstein S, Sudsbury RA, Rotstein OD. Relative roles of Na+/H+ exchange and vacuolar-type H+ ATPases in regulating cytoplasmic pH and function in murine peritoneal macrophages. J Cell Physiol 1993; 157:453-460.
Tartakoff AM. Perturbation of vesicular ttaffic with the carboxylic ionophore monensin. Cell 1983; 32:1026-1028.
Thomsen P, Rudenko O, Berezin V, Norrild B. The HPV-16 E5 oncogene and bafilomycin Ai influence cell motility. Biochim Biophys Acta 1999; 1452:285-295.
Vassalli JD, Sappino AP, Belin D. The plasminogen activator/plasmin system. J. Clin. Invest. 1991;88:1067-1072.
Versantvoort CH, Broxterman HJ, Pinedo HM, et al. Energy-dependent processes involved in reduced drug accumulation in multidrug-resistant human lung cancer cell lines without P-glycoprotein expression. Cancer Res 1992; 52:17-23.
Vik SB, Antonio BJ. A mechanism of proton translocation by Fi Fo ATP synthases suggested by double mutants ofthe a subunit. J Biol Chem 1994; 269:30364-30369.
Webb SD, Sherratt JA, Fish RG. Alterations in proteolytic activity at low pH and its association with invasion: a theoretical model. Clin Exp Metastasis 1999; 17:397-407.
White JM. Membrane fusion. Science 1992; 258:917-924.
Wieczorek H, Putzenlechner M, Zeiske W, Klein U. A vacuolar-type proton pump energizes K-f/H+ antiport in an animal plasma membrane. J Biol Chem 1991; 266:15340-15347.
Yagel S, Khokha R, Denhardt DT, Kerbel RS, Parhar RS, Lala PK. Mechanisms of cellular invasiveness: a comparison of amnion invasion in vitro and metastatic behavior in vivo. J Natl Cancer Inst. 1989;81:768-775.
Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y. Bafilomycin Al prevents maturation of autophagic vacuoles by inhibiting ftision between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct. Funct. 1998;23:33-42.
Zhang J, Feng Y, Forgac M. Proton conduction and bafilomycin binding by the Vo domain ofthe coated vesicle V-ATPase. J Biol Chem 1994; 269:23518-23523.
60
Zimniak L, Dittrich P, Gogarten JP, Kibak H, Taiz L. The cDNA sequence ofthe 69-kDa subunit ofthe carrot vacuolar H+-ATPase. Homology to the beta-chain of Fo Fp ATPases. J Biol Chem 1988; 263:9102-9112.
61
PERMISSION TO COPY
In presenting this thesis in partial ftilfillment of die requu-ements for a master's
degree at Texas Tech University or Texas Tech University Health Sciences Center, I
agree that the Library and my major department shall make it freely available for
research purposes. Permission to copy this thesis for scholarly purposes may be
granted by the Director ofthe Library or my major professor. It is understood that
any copymg or publication of this thesis for financial gam shall not be allowed
without my ftirther written permission and that any user may be liable for copyright