(CANCER RESEARCH 36, 2518-2524, July 1976]
Changes in Plasma Membrane Structure Associated with
Malignant Transformation in Human Urinary
Bladder Epithelium1
RonaldS. Weinstein
Department of Pathology, Rush Medical College, and Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612
Summary
Integral membrane proteins are visualized as intramem
brane particles (IMP; also called membrane-associated pan
tides) at the cleaved surfaces of freeze-fractured plasma
membranes. Topographical distributions of the IMP of
urothelial cell membranes in normal human bladder and for
a small series of low-grade noninvasive transitional cell
carcinomas and invasive transitional cell carcinomas are
shown to be significantly different. Using several statistical
methods that test IMP topography vis a vis the random
(Poisson) hypothesis, it is demonstrated that IMP are mildly
aggregated in plasma membranes of normal human urothe
hal cells and that, in noninvasive carcinomas, IMP aggrega
tion is increased. In invasive transitional cell carcinomas,
IMP are statistically nonaggregated and are in a random
distribution in the plane of the membrane. IMP numerical
densities are also altered in the course of neoplastic trans
formation. IMP are significantly increased in number in
plasma membranes in human noninvasive transitional cell
carcinomas but are similar to control values in invasive
tumors. Loss of IMP and changes in IMP topography may
be related to tumor invasiveness or they may represent
an epiphenomenon.
It is generally acknowledged that changes in the plasma
membrane might account for the biological behavior of
tumors (7, 13, 24, 29). However, none of the many types of
changes that have been described in tumor cell membranes
have been correlated successfully with stages of neoplastic
transformation. In the current study, normal and malignant
urinary bladder epithelia are examined by thin section and
freeze-fracture electron microscopy to determine whether
membrane ultrastructure is modified during neoplastic
transformation and if such structural changes can be come
lated with tumor invasiveness. Particular attention is di
nected to the numerical density and 2-dimensional topogna
phy of IMP,2 a distinctive intramembrane component that
can be visualized by freeze-fracture electron microscopy. A
substantial body of evidence indicates that IMP represent
integral membrane proteins (4, 12, 36) and that their topo
graphical distribution can be modulated by biological con
trol mechanisms (5, 14). In this report, IMP numerical densi
ties and topographical distributions are shown to be sign ifi
cantly different for a small series of noninvasive transitional
cell carcinomas and invasive transitional cell carcinomas
arising spontaneously in human urinary bladder. These ob
servations suggest that alterations within the plasma mem
brane may be related to changes in the biological behavior
of bladder carcinomas.
Introduction
Materials and Methods
Human tumors arising in epithelia may pass through non
invasive stages of development in the course of neoplastic
transformation (2, 8). Recent evidence suggests that intnin
sic tumor cell properties may partly determine the biological
behavior of tumors (9, 15). If intrinsic cell changes are
essential for the acquisition of the property of invasiveness,
then it is reasonable to anticipate that such changes might
first appear when tumors pass from a noninvasive to an
invasive stage of tumor development. Previous efforts to
demonstrate specific structural differences between cells
from invasive carcinomas and cells from tumors at earlier
stages of tumor development have been unsuccessful (18,
19).
@
Presented at the Conference “Early
Lesions and the Development of
Epithelial Cancer.―October 21 to 23, 1975, Bethesda, Md. This work was
supported by Grant CA-14447 from the National Cancer Institute, NIH. and by
United States Air Force Contract F33615-75-R-5001 from the Aerospace
Medical Research Laboratory, Wright-Patterson Air Force Base, Ohio. Early
phases of this work were performed in the Department of Pathology, Tufts
University School of Medicine, Boston, Mass.
2518
Human Urinary Bladder Specimens. Biopsies of human
urinary bladder were obtained through a cystoscope and
fixed immediately (less than 45 sec) for light and electron
microscopy. Histopathological evaluations of biopsies were
performed by light microscopy. Biopsy specimens were
classified into one of 3 categories: Category 1, controls;
Category 2, noninvasive transitional cell carcinoma; or Cat
egory
3,
invasive
transitional
cell
carcinoma.
Tumors
were
classified as “noninvasive―
if histopathological evaluation
of many hematoxylin and eosin-stained paraffin sections
failed to demonstrate penetration of the unothelial base
ment membrane by tumor cells, or as “
invasive―if tumor
clearly extended through the basement membrane. Cases
for which staging was uncertain were eliminated from this
study. Accepted for the study were biopsies from 5 patients
2 The
abbreviations
used
are:
IMP,
intramembrane
particles;
CD.
, coeffi
cient of dispersion; IMPPand IMPE intramembrane particles on fracture face
P and face E. respectively.
CANCER
RESEARCH
VOL. 36
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Integral Membrane Proteins in Cancer
with relatively low-grade noninvasive transitional cell carci
noma, 4 patients with relatively low-grade invasive transi
tional cell carcinoma, and 4 controls. The controls were
from 1 patient with incomplete urethral obstruction, 2 pa
tients with benign prostatic hypertrophy, and from an area
of normal-appearing mucosa from 1 patient with transi
tional cell carcinoma elsewhere in the bladder. It was shown
by both light and electron microscopy that the biopsy speci
mens from all of the controls had normal-appearing bladder
mucosa.
Thin-Section Electron Microscopy. Specimens for thin
section and freeze-fracture electron microscopy were di
vided into 1- to 2-cu mm blocks and were fixed in the
operating room, immediately after surgical removal, either
in cold 2% glutaraldehyde in 0.1 M cacodylate buffer, pH
7.3, or in half-strength Kamnovsky's paraformaldehyde for 1
hr (19). For thin-section electron microscopy, tissues were
postfixed in 1% osmic acid in cacodylate buffer, pH 7.3,
dehydrated by serial passage through graded ethanol solu
tions, embedded in Epon 812, sectioned with diamond
knives, and stained with unanyl acetate and lead citrate.
Thin sections and freeze-fracture replicas were photo
graphed in a Philips EM 300 electron microscope.
Freeze-FractureElectronMicroscopy.Two% glutaralde
hyde-fixed tissue blocks were soaked overnight in a cry
oprotectant consisting of Millonig's phosphate buffer, pH
7.4, containing 20% glycerol (v/v). Blocks were quenched to
_1500
in liquid
Freon
22,
cooled
by liquid
nitrogen,
and
freeze-fractured (22) with a Balzers BAF 301 freeze-etch unit
equipped with an electron beam evaporation device (EVM
052) and a QSC 201 quartz crystal thin-film-thickness moni
ton.
For control urothelium, freeze-fracture replicas of plasma
membranes from regions of intermediate cell-intermediate
cell apposition, intermediate cell-basal cell apposition, and
basal cell-basal cell apposition were photographed. Lu
minal membranes and basal cell membranes, at the stromal
front, were excluded. For tumors, sampling was restricted
to plasma membranes in regions of tumor cell-tumor cell
apposition. Areas of membrane containing cell-cell junc
tions were avoided in both controls and tumors.
Statistical Evaluation of IMP Topography Vis-Ã -VIsthe
Random(Poisson)Hypothesis.If the IMP are truely ran
domly distributed over the membrane fracture face, then
the freqeuncy distribution of IMP would be expected to be
Poisson distributed . Two statistical tests were used to eval
uate IMP topography vis-á-visthe Poisson hypothesis. First,
the C.D. was used as an index of IMP ordering. This repre
sents
the
first
use
of
the
C.D.
statistic
for
the
analysis
of
membrane topography. C.D. is defined as variance/mean
for a frequency distribution curve (32). The statistic is an
indicator of degree of departure of IMP distributions from a
true random distribution. A characteristic of any Poisson
distribution is that C.D. = 1, and any order in the distnibu
tion of IMP would be reflected in the deviation of C.D.
values from unity: distribution of IMP in a lattice-like config
uration causes the C.D. to go below 1, and aggregation of
IMP causes the C.D. to go above 1 (32). Critical values for
the C.D. statistic have not been published. However, it can
be shown that under suitable conditions, such as those in
this study, the C.D. statistic bears a simple relationship to
x2, being distributed
approximately
as
@2/(k—1) for k de
grees of freedom where k is the number of domains (i.e.,
squares in the test lattice).3 As calculated from values in
standard x2 tables, C.D. values over 1.372 are indicative of
statistically significant IMP aggregation (p < 0.01) for a 100square lattice, since C.D. = @@,99/k
—1 = 135.81/99. C.D.
values below 0.708 are indicative of statistically significant
ordering into a lattice-like arrangement (p < 0.01), since
C.D. = [email protected]/k 1 = 70.06/99. C.D. values within the 0.708
to 1.372 range indicate statistical randomness (p < 0.01).
Through computer Monte Carlo simulations, we confirmed
that C.D. values are directly related to topography and
independent of IMP numerical densities under conditions
encountered in this study.
Second, the frequency distribution curve for each test
field was also compared with Poisson distribution by the
variance test for goodness of fit of the Poisson distribution
(28) and classified as Poisson on non-Poisson at p < 0.01.
The percentage of test fields fitting a Poisson distribution is
used as an additional index of prevalence of IMP random
ness for different test fields within categories.
FrequencyDistributionsof IMP. Electronmicrographsof
freeze-fracture replicas were coded and analyses of IMP
topographical distributions were done by a technician,
without knowledge of the clinical history or histopathologi
cal diagnosis for the cases. For statistical analysis of IMP
topographical distributions, electron micnognaphs of nela
tively large flat areas of the PF fracture face (3) of freeze
fracture membrane were printed at a final magnification of
x250,000. IMP frequency distributions were determined by
superimposing a transparency containing a 10-sq cm test
grid (defining a “test
field―),subdivided into 100 1-sq cm
squares, oven individual electron micrographs of freeze
fracture membranes and recording the number of IMP in
each of the 100 squares on a differential counter. Numerical
densities of IMP, defined as the number of IMP per unit area
of membrane, were calculated from the frequency distnibu
tion data. For individual biopsies, topographical distnibu
tions of IMP were determined for 8 to 12 membrane frac
ture faces. EF faces (3) were also evaluated by statistical
methods.
Results
Fine Structureof TransitionalCell Carcinomas.The ul
trastructure of human bladder tumors, as seen by thin
section microscopy, has been described elsewhere (10).
Studies on human bladder with the freeze-fracture tech
nique have been limited to a consideration of cell-cell junc
tions (37). For the purposes of the current study, we exam
med the ultnastructureof normal uroepithelium and of blad
dentumors in order to determine the level of preservation of
the freeze-fractured tissue. In general, tissue preservation
was acceptable (Fig. 1). Myelin figures, swollen mitochon
dna, and dilatedcistemnae,allindicatorsof cellinjury,
were
rarely encountered.
ElectronMicroscopyof Freeze-FracturedHumanUrothe
3 W. D. Selles, R. S. Weinstein,
and I. T. Young. Coefficient
of Dispersion
as a Statistic for Evaluating Cell Membrane Topography, submitted for publi
cation.
JULY 1976
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2519
R. S. Weinstein
hal Membranes. The freeze-fracture process splits open
cell membranes and generates novel fracture faces which
originate from within the interior of the cell membrane (4).
Freeze-fractured urothelial cells in normal bladder and in
tumors are readily recognized by their characteristic sizes
and shapes and by their intercellular relationships. The
epithelial-mesenchymal front can be identified in freeze
fracture replicas for most specimens. For purposes of this
study, analysis of membrane topography was restricted to
cells above the level of basement membrane in both nonin
vasive and invasive transitional cell carcinomas.
Two novel fracture faces are generated by freeze-f nactun
ing cell membranes: “
Face PF―has been defined as the
fracture face of the inner lamella of the membrane, and
“
Face
EF,―
as
the
fracture
face
of
the
outer
lamella
of
4 In
earlier
publications
(20,
2520
36),
the
PF-face
was
called
the
A-face
and
fracture face
(IMP@)Controls
Category
the
EF fracture face
(lMP@)
690―
(95-115)Noninvasive
(530-85O)'@
Carcinoma
(115-160)Invasive
1300
(880-1730)
105
125
530
the
membrane (3). Both fracture faces bear populations of IMP
that average 7 nm in replica diameter and resemble the IMP
which are membrane components of many types of cells (4).
Henceforth, the Face PF IMP shall be designated IMP1.and
the Face EF IMP shall be designated IMP@:
.@
The numerical densities of IMPE:in controls and tumors
were not significantly different (data not included). Analysis
of numerical densities of [email protected] noninvasive transitional
cell carcinomas shows that these values were well above
control values (p < 0.01). In invasive tumors, numerical
densities of IMP,. were not significantly different from con
trol values (Table 1) but were significantly below values for
noninvasive tumors (p < 0.01).
By visual inspection, IMP,. appeared to be similarly dis
tnibuted in the plane of the membrane in both controls (Fig.
2) and in invasive transitional cell carcinomas in which the
IMP appeared to be either randomly distributed or slightly
aggregated (Fig. 4). IMP1. appeared more aggregated in
noninvasive tumors (Fig. 3), although it is noteworthy that
differences in IMP, clustering between noninvasive tumors,
invasive tumors, and controls were not striking to the eye.
IMP@appeared, by visual inspection, to be randomly distnib
uted for specimens in all 3 categories.
C.D. Values. Analysis of topographical distributions of
IMP,. by means of the CD. statistic showed significant dif
fenences among all 3 groups (Table 2). The mean CD. value
(±S.D)for controls, 1.54 ±0.072, is indicative of mild IMP
aggregation. The mean CD. values for noninvasive transi
tional cell carcinomas, 1.82 ±0.110, was higher than con
trol values, indicating enhanced 1MP1 aggregation. The
mean CD. value for invasive carcinomas, 1.26 ±0.061,
indicates that the IMP,. are within the range that is indicative
of a truly random distribution. All 5 noninvasive transitional
cell carcinomas had C.D. values higher than the control
values. The percentage of test fields fitting a Poisson hy
pothesis is highest for invasive carcinoma and lowest for
noninvasive carcinomas, with controls showing an intenme
diate level of conformity to the Poisson distribution (Table
2).
Comparisons of Populations of Cells. There can be con
siderable variability of IMP topographical distribution from
cell to cell within a single biopsy specimen, although the
“average―
distributions are similar from biopsy to biopsy
EF-face was called the B-face.
Table1
Numericaldensitiesof IMP within freeze-fracturedcell membranes
in nonneoplastic human urothelium and in transitional cell
carcinomas
IMP counts were made on 10 PF and EF faces for each biopsy.
Numbersof patients: controls, 4; noninvasivecarcinoma, 5; inva
sive carcionma, 4.IMP
@m)PF
numerical densities (IMP/sq
Carcinoma
(1 Mean
for
all
100
(400-670)
specimens
within
the
(90-135)
category.
One-way
analysis
of varianceshows that differences in IMPdensities on PFfaces are
significant
(p < 0.01). IMP densities
on EF faces are not signifi
cantly different.
b Number in parentheses,
specimens.
range of mean values for individual
Table 2
Statistical analysis of IMP topographical distributions on PF faces
of freeze-fracturedhuman urothelium
Analyses of EF faces showed no significant differences in the
topographical distributions of IMP. Data are not included for EF
faces.
of dis
CategoryCoefficient
PoissonControls
0.072'50'Noninvasive
(4/44)°1
0.11013.6Invasive
carcinoma (5/48)1.82
carcinoma (4/48)1
a Numbers
in
parentheses:
persion%
.54 ±
±
.26 ±0.06181.8
numerator,
number
of
biopsies;
de
nominator, total number of test fields for all biopsies within the
category.
b Mean ±S.D. CD.
values were compared by 1-way analysis of
variance,showing that there is a highly significant difference in IMP
clustering among the 3 categories (p < 0.01).
C Fit
to
a
Poisson
distribution
was
determined
by
the
variance
test
for goodnessoffit to a Poissondistribution. Acceptanceas Poisson
was atp < 0.01.
within each category. To compare distributions across cate
gonies while accounting for cell-to-cell variability, cumula
tive distributions were generated from data pooled from all
cases within a single category. For each test field, the
variance test for goodness of fit of a Poisson distribution
yields a @2
variable. This variable can be normalized by a
standard formula as follows:
S.D. = (x2
where N = number of degrees of freedom. The cumulative
distribution of normalized x2's is plotted for each category
(Chart 1). The curves for pooled data from controls, nonin
vasive carcinomas, and invasive carcinomas (Chart 1) are
significantly different from one another (p < 0.01 ), as deter
mined by the 2-sample Kolmogorov-Smimnov test, confirm
ing that the test fields are drawn from different membrane
populations.
CANCER RESEARCHVOL. 36
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Integral Membrane Proteins in Cancer
STANOARL'
0EV/AT/OW(units fromw,ecnl
Chart 1. Cumulative relative frequencies of normalized @2
values for the
topographical distributions of IMP in test fields of freeze-fractured human
urinary bladder cell membranes. The @2
values were generated by the van
ance test for goodness offit of the Poisson distribution and then normalized.
The normalization is: S.D. = It(x2 —N)/@/@Np(,
where N = the number of
degrees of freedom. In this instance, N was determined from the number of
classes of IMP, n, where: n = t(maximum number of IMP/test square) + 11.
Numbers of test fields within categories represented by the curves are:
invasive carcinomas, 48, controls, 44; and noninvasive carcinomas, 48. IMP
topographical randomness is closely approximated in invasive transitional
cell carcinomas, and IMP aggregation is greatest in noninvasive transitional
cell carcinomas.
Discussion
Integral membrane proteins are defined as amphiphatic
proteins that span the lipid bilayer region of the cell mem
brane (31). They are a heterogenous group of molecules
that may participate in a number of cellular processes in
cluding membrane transport, transmembrane coupling,
and cell-cell recognition (16, 21, 26, 30). A substantial body
of evidence supports the hypothesis that the IMP visualized
by freeze-fracture electron microscopy are proteins or pro
tein aggregates intercalated into the membrane lipid bilayer
(4, 12, 34). Changes in the numerical densities (35) and
topographical distributions of IMP (1, 30) have been ob
served in association with transformation in several tissue
culture systems but have not been previously reported in
solid tissues undergoing neoplastic transformation.
The topographical distributions of some IMP may be in
fluenced by interactions of the IMP with either other integral
membrane components (1, 33) or elements of the cell cyto
skeleton, including peripheral membrane proteins (6, 23),
which may place constraints upon the lateral mobility of
IMP. Theoretically, other IMP could be free of these con
straints and therefore be capable of achieving truly random
distributions in diffusion fields within the membrane. Alter
ations in the topographical distributions of IMP can be
induced by both physiological (5, 14) and unphysiological
(11, 17, 25, 33) methods. This multiplicity of factors that may
influence topography enormously complicates the task of
ascribing changes in IMP topography in pathological states
to specific mechanisms.
In this quantitative electron microscopy study on human
bladder urothelia, we explored the possibility that changes
in numbers of IMP and in the topography of IMP may serve
as markers for specific stages in neoplastic transformation.
We found that numbers of IMP are increased above control
levels in noninvasive transitional cell carcinomas but me
duced in invasive transitional cell carcinomas. In addition,
there are significant differences in the topographical distri
butions of IMP between noninvasive and invasive human
carcinomas. IMP. are mildly aggregated in nonneoplastic
epithelia. IMP. aggregation is enhanced in noninvasive
transitional cell carcinomas whereas, in invasive cancino
mas, IMP. are randomly distributed in the large majority of
tumor cells.
The molecular mechanisms involved in producing the
dramatic increases in IMP1.numerical densities in noninva
sive transitional cell carcinomas are unknown. High densi
ties of IMP have been associated with high levels of mem
brane metabolic activity (4). However, there is no reason to
believe that the level of metabolic activity of plasma mem
branes of noninvasive tumors is greater than that of invasive
tumors of the same histopathological grade. Increased IMP
densities also have been observed in plasma membranes of
cells transformed with oncogenic viruses (35), although
such changes do not invariably accompany transformation
(27, 30).
There are a number of plausible explanations for the
differences in IMP1.topography observed between controls
and tumors. One possibility is that artifacts are introduced
at cystoscopy, a technique which requires infusion of un
physiological solutions, such as filtered water and/or hypo
tonic glycine, into the bladder. Normal unothelium and tran
sitional cell carcinomas differ with respect to gross and
microscopic morphology, leakiness to bypass diffusion,
vasculanity, and stroma. Because of these differences, anti
factual alterations in IMP may be preferentially introduced
into certain categories of urothelial cells. The possibility of
cystoscopy artifacts is currently being systematically exam
med in this laboratory, and the preliminary data show that
significant ultrastructural artifacts can be induced by con
ventional cystoscopy procedures (B. V. Pauli, R. S. Wein
stein, and J. Alnoy, unpublished data).
Another possibility, not necessarily unrelated to the first,
is that differences in IMP1.topography can be explained on
the basis of IMP.-cytoskeleton interrelationships. In normal
cells, the topographical distributions of some IMP may be
regulated by peripheral membrane proteins, elements of the
cell cytoskeleton, to which the IMP1. may be bound at the
juxtacytoplasmic surface of the plasma membrane. We sug
gest that, in noninvasive transitional cell carcinomas, alter
ations in the cytoskeleton may produce a redistribution of
IMP, . Changes in the cytoskeleton could result either from a
primary structural modification of the cytoskeleton (15) or
from changes in cell shape produced by other etiologies
(16). Although a mechanism involving cytoskeletal pentuba
tions with maintenance of IMP-cytoskeleton binding could
account for enhanced IMP1@
aggregation in noninvasive tu
moms,it is unlikely that this Would account for the random
distributions of 1MP1in the plasma membranes of invasive
tumor cells. Random distributions could be accounted for
on the basis of inadequate binding of IMP,. to the cytoskele
ton. This would relieve IMP1.of constraints upon their lateral
mobility and permit IMP1@
to achieve truly random distnibu
tions within the plane of the membrane. Based upon these
observations, it is postulated that loss of control of IMP@
topography may represent a relatively late step in neoplastic
transformation.
JULY 1976
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2521
R. S. Weinstein
Acknowledgments
The author gratefully acknowledges the expert technical assistance of
Jonathan S. Wallach and William Leonard. I also take pleasure in thanking
Dr. Ian T. Young, Massachusetts Institute of Technology, Cambridge, Mass..,.
and William D. SelIes, New England Medical Center Hospital, Boston, Mass.,
for suggestir@gthe statistical tests used in this study and for assisting in the
evaluation of the data.
19.
20.
21.
22.
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Fig. 1. Freeze-fracture replica of a tumor cell in a Grade 1 human noninvasive transitional cell carcinoma. PM, plasma membrane; mit, mitochondnia;
mvb, multivesicular body. Arrows, position of nuclear pores within the nuclear membrane. x 36,200.
Fig. 2. PF and EF fracture faces of plasma membranes of 2 unothelial cells in normal human urinary bladder. The PF face bears many more IMP than the EF
face. The IMPPappear to be mildly aggregated. ECS, extracellular space. x 75,000.
Fig. 3. PF and EF fracture faces of plasma membranes in a Grade 1 noninvasive transitional cell carcinoma. IMPPare increased in numerical density and
show a tendency to aggregate. x 80,000.
2522
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CANCER RESEARCHVOL. 36
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Changes in Plasma Membrane Structure Associated with
Malignant Transformation in Human Urinary Bladder Epithelium
Ronald S. Weinstein
Cancer Res 1976;36:2518-2524.
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