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Rat adrenocortical carcinoma 494An integrated structural stereological and biochemical analysis.

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Rat Adrenocortical Carcinoma 494: An Integrated Structural,
Stereological, and Biochemical Analysis'
ROBERT N. MOORE,* DAVID P. PENNEY AND KATHY A. AVERILL
Departments ofAnatomy and Dental Research, University of Rochester, School of
Medicine and Dentrstry, Rochester, New York 14642
ABSTRACT
Snell adrenocortical tumor 494 was implanted into male
Sprague-Dawley rats and recovered 7 , 1 4 , 2 1 , 2 8or 35 days following initial detection by palpation (7-10 days following transplantation). Electron microscopic,
stereologic and biochemical analyses of the tumor were compared to adrenals of
normal animals to serve as a baseline for further studies of the effects of chemotherapeutic agents on tumor cells. Tumor cells possessed oval or elongated mitochondrial profiles with tubular cristae, one or two very large (> 5 p m ) lipid droplets, abundant ribosomes and coated vesicles, and sparse rough and smooth endoplasmic reticulum. Stereologic evaluation revealed t h a t tumor lipid volume was
41% and mitochondrial volume 29% t h a t of t h e normal adrenal controls. Tumor
nuclei were 2.5 times larger than adrenocortical nuclei while cellular volumes
were similar. On a net weight basis, tumor cholesterol was 55%, cholesterol ester
2.2%, and lipid phosphate 25% of respective mean values for normal adrenal
glands. The tumor cholesterol: cholesterol ester ratio progressively decreased
with time, but remained 18-fold greater than the normal adrenal. Plasma corticosterone levels in tumor-bearingrats were elevated %fold by 14 days after initial detection. The adrenals of t h e tumor-bearing host exhibited marked involution, t h e extent of which was directly related to tumor size.
The Snell transplantable rat adrenocortical
carcinoma 494 h e l l and Stewart, '59) is a n
excellent model for the study of adrenocortical
neoplasia in t h a t i t was of spontaneous origin,
is relatively insensitive to ACTH (Kimmel e t
al., '74; Ney et al., '691, and is corticosteronesecreting (Sharma, '73; Sharma and Brush,
'73). For these reasons, this tumor resembles
many human adrenocortical tumors (Symington, '69). Kimmel e t al. ('74) have described
large clear vesicles and deficiencies in t h e
number and internal structure of mitochondria in transplanted tumor cells grown subcutaneously for four to six weeks. Isolated
tumor cell preparations exhibit sparse numbers of lipid droplets and mitochondria, and
moderately well developed smooth endoplasmic reticulum and Golgi complexes (Sharma
and Hashimoto, '72). When compared to t h e
normal adrenal gland, this tumor has been
shown to be 1-10% as efficient in corticosterone production (Ney et al., '69) and has decreased ll@-hydroxylaseand 5a-reductase activities (Johnson e t al., '61; Kimmel et al., '74;
Maynard and Cameron, '72; Peron et al., '74;
ANAT. REC. (1978) 190: 703-718.
Sweat and Bryson, '64). Energy (ATP) for
rapid tumor cell proliferation may be in part
provided by a n a-glycerol phosphate shuttle
which compensates for the markedly decreased ability of tumor cells to utilize glucose
for pyruvate and lactate production (Peron et
al., '74). Although tumor adenylate cyclase
was similar in activity and content to t h a t of
t h e normal adrenal (Ney e t al., '691, its stimulation by epinephrine, norepinephrine and
TSH suggested the specificity of t h e membrane receptor to ACTH had been modified,
and could now be activated by other hormones
(Schorr and Ney, '71; Schorr et al., '71).
Adrenal glands in female rats bearing the
tumor were atrophic and characterized by
significant decreases in t h e number and surface area of internal mitochondrial vesicles
(Nickerson, '73). Mitochondria were elongated
in shape rather t h a n spherical and the volume
Received Oct. 22. '76. Accepted Sept. 29, '77.
I Supported by U.S.P.H.S. Grants CA 11198 and DE-00003 and
American Cancer Society Grant IN-1BN.
Present address: School of Dentistry, University of California, Los
Angeles, California 90024.
To whom reprint requests should be addressed.
703
704
ROBERT N. MOORE, DAVID P. PENNEY AND KATHY A. AVERILL
of mitochondria per cell was reduced. A significant decrease in the volume and surface area
of smooth endoplasmic reticulum was also observed. These morphological manifestations of
suppressed adrenocortical function were a t tributed to the elevated blood levels of corticosteroids produced by the tumor which
negatively feeds back on the hypothalamo-hypophyseal axis and decreases ACTH release.
Although certain morphologic and biochemical aspects of this tumor have been previously
reported, there has been no integrated study
utilizing such parameters in a specific temporal sequence following transplantation. I t
will be the purpose of this investigation to
ascertain on a quantitative basis the interrelationships of fine structure and lipid biochemistry of adrenocortical carcinoma 494
cells during their establishment and growth
following implantation. In addition to providing further information to aid in the characterization of the tumor, these data will serve
as a baseline to which further studies involving the effects of clinically-utilized chemotherapeutic agents on tumor growth and metabolism will be compared.
MATERIALS AND METHODS
weighed. Specimens of the tumor and adrenals
were taken for fine structural and biochemical analyses, and trunk blood was collected
for plasma corticosterone determinations.
Electron microscopy
Pieces of non-necrotic viable tumor, and
normal adrenal were fixed in formaldehydeglutaraldehyde (Karnovsky, '65) for three
hours, rinsed overnight in 0.1 M sodium
cacodylate buffer (pH 7.21, post-fixed for one
hour in 1%OsO,, dehydrated in increasing
concentrations of ethanol, and embedded in
epoxy resin (Spurr, '69). Thin sections (40-80
nm) were stained with uranyl acetate and lead
citrate and observed and photographed with
an RCA 3-H electron microscope.
Stereology
Tumor and adrenocortical tissues were
taken from each animal in each experimental group and processed as noted above.
Stereologic data was obtained by the differential point counting method of Weibel ('69)
using a Plexiglas grid apparatus (Moore et al.,
'77). From each group, three tissue blocks
were selected at random and a t least 50 electron micrographs were taken of the upper
right hand corners of the grid openings a t a
constant magnification of 3,100 X . These
plates were then printed to a standard magnification of 13,330 X . The nuclear fraction
was obtained directly from differential point
counting while the cytoplasmic volume fraction was determined by subtracting the sum of
the extracellular space, nucleus, and necrotic
tissue fractions from the total volume.
Male albino Sprague-Dawley rats weighing
80-100 g a t the start of the experimentation
were housed in an air conditioned room and
exposed to a 12:12hour 1ight:dark cycle (6:OO
A.M. to 6:OO P.M.). Purina Laboratory Chow
and fresh drinking water were supplied ad
libitum. The carcinoma was obtained from
tumor-bearing hosts maintained in the laboratory. Pieces of viable tumor approximately 2
mm3 in size were transplanted subcutaneousBiochemistry
ly via trocar into the dorsolumbar region of
ether-anesthetized rats. When the implant
Lipids were separated from tumor and
became palpable, usually 5-10 mm in cutane- adrenal samples by homogenization in methously-measureddiameter by seven to ten days, anol followed by subsequent extraction in
the tumors were measured along their long chloroform: methanol (1:1) (Sperry and
and short axes with a caliper and their mean Brand, ' 5 5 ) . Lipid fractionation was achieved
diameters calculated.
by thin layer chromatography using silica gel
To assess the influences of the growing plates (EM Laboratories) which were detumor on the host animal, daily body weights veloped in petroleum ether:diethyl ether:aceand tumor diameters (2 axes) were recorded. tic acid (9O:lO:l) (Skipski and Barclay, '69).
Animals (10-15 per group) were sacrificed by Concentrations of cholesterol and cholesterol
decapitation 7,14,21,28and 35 days after the esters were determined colorimetrically using
tumors became palpable. Normal animals (5-6 the Liebermann-Burchard procedure employper group) of equivalent age served as con- ing acetic anhydride, acetic acid and sulfuric
trols. At sacrifice (10:00-11:00A.M. for each acid (86:82:25) (Kabara, '62). The phosphate
designated time period), the tumors and nor- content of the lipid fraction was analyzed by
mal adrenals were removed, measured, and the method of Lindberg and Ernstner ('56).
STRUCTURE AND FUNCTION OF RAT ADRENOCORTICAL TUMOR
705
400
CONTROL
TUMOR-BEARING
A TUMOR SIZE
7;- 350-
0
e
7=. 300-
s
&
k
$
250TUMOR
PALPABLE
200-
a'
Q
/
G
3
150-
2
50-
B
50 -
i
a
0
I
0
5
I
10
1
15
20
25
30
5
TUMOR WEIGHT (gm)
Fig. 2 Relationship of total adrenal weight to adrenocortical tumor weight in male Sprague-Dawley rats
Each dot represents one animal.
Plasma free corticosterone was measured by
t h e fluorimetric method of Nielsen and
Asfeldt ('67).
RESULTS
Effect of tumor on body and adrenal weights
The body weights of tumor-bearing animals
were similar to those of control animals until
day 30, after which the rate of body weight
gain diminished in tumor-bearing animals
(fig. 1).Thus, the presence of a functional
tumor did not appear to evoke any marked
deleterious effects on general body growth
until the tumor had attained considerable size
(> 25 mm mean diameter). Similarly the absolute adrenal weight was inversely related to
the weight of the tumor (fig. 21, with marked
adrenal atrophy being present in animals with
tumors that exceeded 4 g (> 25 mm mean
diameter).
706
ROBERT N. MOORE, DAVID P. PENNEY AND KATHY A. AVERILL
Tumor morphology
Tumors of all stages of growth were composed of cells in close approximation to sinusoids. Giant multinucleated or polyploid cells,
which are present in many carcinomas, were
not observed. Cells, which morphologically appeared to be degenerative, were found occasionally interspersed among the viable cells.
Degenerative cells were more frequently observed farther from the vessel lumen, especially in tumors of larger diameters, suggesting a n ischemic etiology. In addition, some
cells were characterized by less electron dense
mitochondrial and cytoplasmic matrices, loss
of mitochondrial cristae, distended nuclear
membranes, vacuoles or enlarged vesicles, and
distended cisternae of t h e endoplasmic reticulum (figs. 5, 6). Cells exhibiting such signs of
degeneration were considered to be at a n intermediate stage in the continuum of the degenerative process and will not be specially
characterized. Thus, this tumor appeared to be
composed of only one basic tumor cell type.
The following morphological characterization
will be restricted t o viable tumor cells. The 7day tumor will be described and age-related
changes will be subsequently noted.
Nuclei were mostly round to ovoid, not
indented, predominantly euchromatic a n d
usually possessed one to two nucleoli. I n contrast to normal adrenocortical mitochondria
of the zona fasciculata (fig. 61, which have a
rounded profile and a vesicular internal structure, tumor cell mitochondria were smaller,
less numerous, and were frequently elongated
or pleomorphic in shape (fig. 4). The sparse intramitochondrial membranes were predominantly long and tubular (characteristic of
cells of the adrenal zona glomerulosa) or occasionally small and vesicular (characteristic of
cells of the zona fasciculata). Intracellular
lipid was not as dispersed in the tumor as in
the normal adrenal cortex, being frequently
aggregated into one or two extremely large
(some > 5 p m in diameter) lipid droplets (fig.
5). In addition to varying amounts of rough
endoplasmic reticulum (RER), which is rarely
observed in normal rat adrenocortical cells,
tumor cells contained numerous polysomes
and ribosomes arranged either in rosette patterns or free in the cytoplasm (fig. 4). In
marked contrast to normal adrenocortical
cells (fig. 61, the smooth endoplasmic reticulum (SER) was poorly defined and was usually
noted only in small, scattered areas of the cy-
toplasm (fig. 4). Distended cisternae of either
t h e SER or RER were infrequently encountered in viable tumor cells. The Golgi complex
and stacks of straight, parallel profiles most
likely of Golgi origin were frequently observed
and occasionally extended for considerable
distances in the cytoplasm (fig. 4).Coated vesicles (fig. 5) and lysosomal dense bodies,
bounded by a single limiting membrane, were
noted (fig. 81, although their presence from
cell to cell was variable. Microvilli were regularly found extending into the intercellular
space (fig. 8). Contacts between viable cells
(fig. 10) were relatively common, and when
observed, were usually represented by small
gap junctions, rather than desmosomes. The
tumor was moderately vascularized, the capillary endothelium was fenestrated, and the
surrounding basement membrane was essentially intact. No evidence of viral particles
was observed.
While cells with the above morphologic features were present for all time periods investigated, with advancing age, most tumors
showed a general trend toward a n increase in
both the size of the lipid droplets and their
juxtapositioning to profiles of the smooth endoplasmic reticulum (fig. 9) Although the endoplasmic reticulum remained poorly developed throughout all time periods, there appeared to be a gradual shift from the rough to
the smooth variety with increasing posttransplantation time (fig. 9). Mitotic activity
remained prominent, despite the considerable
size of the tumor. Intercellular spaces were
larger, and cells exhibiting degenerative characteristics were more widespread, again suggesting t h a t the tumor was outgrowing its
blood supply.
Stereolog3
In the normal adrenal cortex, necrotic tissue is rarely seen. The tumor frequently contained necrotic cells and thus had a greater
percentage for all experimental periods (table
1). The mean volume fraction of tumor cell nuclei from all age groups was 2.5 times greater
(range: 2.3-2.8 x 1 than adrenal nuclei. Therefore, since the total parenchymal cell volume
of t h e tumor was similar to that of the adrenal cortex, the cytoplasmic volume (cellular
minus nuclear volumes) of the tumor was correspondingly less. The volume fractions of mitochondria and lipid of the tumor averaged
29% (range: 21-38%) and 41% (range: 3071x1, respectively, those of the normal adre-
Cell = Total volume - (extracellular and necrotic).
nucleus.
Cytoplasm = Cell
~
0
O.lf0.1
0.7-t 0.1
0.7-t 0.8
13.12 1.4
5.32 0.8
42.92 1.5
16.1k0.9
9.42 1.3
22.4-t 2.3
80.62 1.4
63.32 1.2
10.0%1.3
13.520.8
30.0-t 1.3
86.32 1.8
0.320.1
0
0.220.2
0
0.120.1
0.62 0.1
16.5-t 1.9
4.3% 1.0
42.42 1.8
13.72 0.6
9.9? 1.1
23.82 1.7
82.l-t 1.3
57.82 1.5
92.02 1.2
81.62 1.6
8.02 1.3
17.22 1.1
0
1.22 0.7
0
0.320.1
0.82 0.2
0.720.1
5.120.6
3.62 1.0
50.622.2
12.62 0.6
11.4-t 1.4
26.22 2.3
78.02 1.5
57.62 1.8
0.42 0.4
8.72 2.3
10.22 1.4
7.52 1.0
89.82 1.4
83.82 1.6
0
0.42 0.1
0.6-t 0.1
0
0.620.2
0 . 8 2 0.2
8.7% 1.2
24.4% 1.4
86.12 1.5
90.12 1.3
0
1.32 0.6
13.9% 1.8
8.620.7
77.4% 1.7
65.7k 2.1
81.3% 1.8
55.42 1.6
91.12 1.7
80.42 1.7
0
8 . 3 2 1.8
8.92 1.8
11.32 1.5
49.522.0
10.5%0.6
7.12 1.0
2.52 0.5
Lipid
41.222.0
13.520.6
Mitochondria
0.1-t 0.3
0.620.1
Nucleus
Secondary
lysosomes
(auto + l o r
heterophagosomes)
11.72 1.2
3.62 1.0
Cytoplasm
9.8-f 0.6
25.02 2.1
Cell
Lysosomal
dense
bodies
Necrotic
cells and
tissue
' Days following initial tumor palpation or equivalent in normal.
Adrenal cortex
Tumor 494
11. 14days
Adrenal cortex
Tumor 494
111.
21 days
Adrenal cortex
Tumor 494
IV.
28 days
Adrenal cortex
Tumor 494
V. 35days
Adrenal cortex
Tumor 494
I. 7 days
Experimental
group '
Extra cellular
space and
non-parenchymal
cells
S.E.)
%
Volume fraction of cells and organelles of normal rat adrenal cortex and adrenocortical tumor 494 (mean percentage
TABLE 1
4
4
0
708
ROBERT N. MOORE, DAVID P. PENNEY AND KATHY A. AVERILL
TABLE 2
Biochemical analyses of normal rat adrenal gland and adrenocortical tumor 494
(mail00 mg wet tissue weight, mean t S.E.)
~~~~
~~
Experimental
erouv
I. 7 days
Adrenal
Tumor
11. 14days
Adrenal
Tumor
111. 21 days
Adrenal
Tumor
I V . 28days
Adrenal
Tumor
V. 35 days
Adrenal
Tumor
' Days following
p
<
Cholesterol
esters (CE)
Ratio
C/CE
Lipid
phosphate
13.59-1-0.25
0.085 0.01
0.13-tO.01
8.22-1-1.07
0.22'0.01
0 07-tO.01
1.18*0.18
0.732 0.04
14.22' 1.16
0.18-t 0.02
0.08' 0.02
4.632 0.54
0.25-t 0.02
0.0720.01
1.4820.13
1.06-tO.08
16.93' 2.28
0.50-t 0.04
0.095 0.01
2.21'0.26
0.29' 0.03
0.08-t 0.01
1.21-tO.08
0.58-t 0.04
11.85-tO.07
0.322 0.03
0.102 0.01
1.8820.15
0.23-tO.01
0.06-tO.01
1.33'0.24
0.7920.06
14.402 0.55
0.535 0.06
0.09-t 0.01
1.59' 0.16
0.17+-0.01
0.032 0.01
Cholesterol
IC)
1.745 0.03
0.60*0.02
'
initial tumor palpation or equivalent in normal
0.001 for all values.
+
7
--&
14
EXPERlr' NTAL PERIOD IN DAYS
Fig. 3 Plasma corticosterone levels of control and tumor-bearing rats. Each bar represents the means of six
to eight animals 2 SEM.
nal. Volume fractions of lysosomal dense
bodies were similar in both tissues. Secondary
lysosomes, which were infrequently observed
in the normal adrenocortical cells, accounted
for a small volume fraction (0.1-0.6%) in
tumor cells.
Biochemistry
The lipid content of the tumor differed
markedly from the normal adrenal gland
(table 2). Cholesterol content averaged 55%
(range: 34-72%) and cholesterol ester levels
2.2% (range: 0.6-3.7%)those of the normal
adrenal gland. With time there was a progressive decrease in the ratio of cholesterol:
cholesterol ester in the tumor. At seven days,
the tumor had an average of eight times more
cholesterol than cholesterol esters. At 35 days
the ratio had decreased to 1.6 times, but remained 18-fold greater than for normal ad-
STRUCTURE AND FUNCTION OF RAT ADRENOCORTICAL TUMOR
renal glands. Lipid phosphate was relatively
consistent in all time groups and averaged
25% t h a t of the normal adrenal. Although
somewhat variable, plasma corticosterone
levels of tumor-bearing rats were elevated, as
much as %fold over normal control values (fig.
31, confirming t h e observations of Ney e t al.
('69).
709
degradation (e.g., nucleolemma distention,
RER and SER dilation) and were therefore
considered to represent degenerated or degenerating cells. As noted earlier, tumor cells
have been shown to be ACTH-insensitive (Ney
e t al., '69). Since Kahri et al. ('70) have shown
t h e maintenance of the vesicular interna of
mitochondria of the zona fasciculata to be
dependent on available ACTH, the refractility
DISCUSSION
of tumor cells to ACTH may be directly reThe organellar fine structure of the car- sponsible for the lamellar configuration of the
cinoma cells exhibited considerable variabil- intramitochondrial membranes.
ity when compared to the organelles of t h e
The marked deviation of tumor cells from
adrenocortices of normal animals. The much normal adrenocortical morphology is in the
larger (2.5 times) nuclear volume fraction is drastic reduction in number and increase in
consistent with the concept of a very rapidly size of lipid dropets. Such changes are usually
metabolizing, aggressive neoplastic cell. The reflective of decreased hormone biosynthesis
most striking deviations from normal occur in (Greep and Deane, '49). In normal steroid-sethe cytoplasmic organelles, especially mito- creting tissues, these lipid droplets are reserchondria, lipid droplets, and endoplasmic re- voirs of hormone precursors, cholesterol, and
ticulum. Similar observations have been re- especially cholesterol esters (Moses e t al., '69).
ported in trypsinized tumor cells (Sharma and However, in the adrenocortical tumor cells
Hashimoto, '72) and in longer-term implants (table 21, t h e opposite is true and lipid, which
is reduced to 41% t h a t of normal adreno(Kimmel e t al., '74).
When non-parenchymal cells, which a r e cortices, is primarily cholesterol, and t h e camore prominent in the tumor, are deleted from pacity to esterify cholesterol appears to be
consideration (table 11, there is no great dif- markedly reduced. Since similar findings were
ference in t h e average amount of extracellu- reported by Lossow e t al. ('65) for t h e 494H
lar space between the two tissues. Because of tumor line, a sub-line of t h e same tumor estabthe tumor's tendency to outgrow its blood sup- lished by several passages through hypophyply, there a r e many more necrotic cells than in sectomized hosts, it appears t h a t the limited
capacity to esterify cholesterol may be inthe normal adrenal (table 1).
Kimmel e t al. ('74) have reported t h a t on a herent in the tumor line and may not be atprotein/tissue net weight basis, there is a 75% tributable solely t o a functional alteration
reduction of mitochondria in tumor cells. The induced by transplantation into hormonallypresent stereologic data (table 1) a r e in close deprived, hypophysectomized animals (494H).
With time, there is a progressive decrease in
agreement and show a 71%reduction in t h e
the cholesterol/cholesterol ester ratios in t u mitochondrial volume fraction.
The diminished numbers of mitochondria mors (table 21, suggestive of a n increasing caand their loss or reduction of internal mem- pacity of cells of more mature and established
branes have been directly correlated with t h e tumors to esterify cholesterol. This phenomeconcomitant reduction of mitochondrial 1 lp- non appears to be directly correlated with t h e
hydroxylase activity in tumor cells (Kimmel gradual increase in smooth endoplasmic retice t al., '74). The deficiency of this enzyme, ulum (SER) of tumor cells with time (fig. 7).
which in the rat controls the conversion of 11- Since it has been proposed t h a t t h e enzymes
deoxycorticosterone (DOC) to corticosterone, involved in cholesterol esterification occur in
t h e principal glucocorticoid synthesized, con- t h e SER, which is never a s well developed in
tributes significantly to the overall inefficien- tumor cells as in normal adrenocortical cells,
cy (< 10%of normal adrenal cortical cells) of t h e efficiency of the tumor in cholesterol
t h e tumor cells in hormone biosynthesis and esterification will most likely remain signifisecretion (Ney e t al., '69). The marked loss of cantly diminished even in longer periods of
mitochondrial matricial density and t h e pres- time post-transplantation.
Lipid phosphate of t h e tumor was about 25%
ence of intramitochondrial myelinated bodies
(Kimmel e t al., '74; Sharma and Hashimoto, t h a t of the normal adrenal cortex. Assuming
'72) were observed in our studies only in those this parameter is predominantly a n index of
tumor cells which also exhibited other signs of cytomembranes, t h e results were in agree-
710
ROBERT N. MOORE, DAVID P. PENNEY AND KATHY A. AVERILL
ment with the fine structural data which
revealed a marked decrease of the ER, both
rough and smooth and mitochondria1 internal
membranes in tumor cells is compared to normal adrenocortical cells.
Generally, the plasma corticosterone values
for tumor-bearing rats (fig. 3) were approximately two to three times greater than controls for the first 21 days following establishment. After this time there was a decrease in
the values almost to control levels, despite
tumor enlargement (fig. l ) ,with presumably a
concomitant increase in hormone production.
This change most likely reflects the marked
adrenal atrophy which is evident in tumorbearing animals by this time (fig. 2 ) . The
corticosterone secreted by t h e tumor is
efficacious in reducing ACTH secretion via
the hypothalamo-hypophyseal-adrenocortical
axis, and the adrenocortices, therefore, resemble glands from hypophysectomized animals.
Since the tumor is essentially insensitive to
ACTH, the reduced secretion of the trophin is
not influential in modifying tumor structure
and function. In addition, no evidence currently exists that tumor hormone secretion follows
any circadian-like pattern as does the adrenal
cortex.
ACKNOWLEDGMENTS
The authors are indebted to Doctor Robert
L. Ney, University of North Carolina, for supplying the adrenocortical carcinoma, to Dr.
Guido V. Marinetti, University of Rochester,
for assistance with the biochemical determinations, and to Mrs. Susan Walker for sectioning the tissue for electron microscopy.
LITERATURE CITED
Greep, R. O., and H. W. Deane 1949 Histological, cytochemical and physiological observations on the pegeneration of the rat's adrenal gland following enucleation. Endocrin., 45: 42-56.
Johnson, D. F., K. C. Snell, D. Francois and E. Heftmann
1961 In Vitro metabolism of p r o g e s t e r o n e 4 ' T in an
adrenocortical carcinoma of the rat. Acta Endocrin., 37:
329-335.
Kabara, J. J . 1962 Determination and microscopic
localization of cholesterol. Methods Biochem. Analysis,
10 263-318.
Kahri, A. R. 1970 Selective inhibition by chloramphenicol of ACTH-induced reorganization of inner mitochondrial membranes in fetal adrenal cortical cells in tissue cultures. Am. J. Anat., 127: 103-130.
Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde
fixative of high osmolarity for use in electron microscopy.
J. Cell Biol., 27: 127A.
Kimmel, G . L., F. G. Peron, A. Haksar, E. Bedigian, W. F.
Robidoux, J r . and M. T. Lin 1974 Ultrastructure,
steroidogenic potential, and energy metabolism of the
Snell adrenocortical carcinoma 494. J. Cell Biol., 62:
152-163.
Lindberg, O., and L. Ernster 1956 Determination of organic
phosphorus compounds by phosphate analysis. Methods
Biochem. Analysis, 3: 1-22.
Lossow, W. J., G. Shyamala, S. Shah and I. L. Chaikoff 1965
Uptake, hydrolosis and synthesis of cholesterol esters by
Exp.
a transplantable adrenal cortical tumor. Proc. SOC.
Biol. Med., 119: 125-131.
Maynard, P. V., and E. H. D. Cameron 1972 Metabolism of
C,,-steroids by homogenates of normal rat and mouse
adrenal tissue and of the Snell transplantable rat adrenocortical tumor 494. Biochem. J., 126: 99-106.
Moore, R. N., D. P. Penney, K. Averill and D. Kurtz 1977 A
modified grid apparatus for stereological analysis of light
and electron micrographs. Acta Anat., 98: 21-23.
Moses, H. L., W. W. Davis, A. S. Rosenthal and L. D. Garren
1969 Adrenal cholesterol: Localization by electron microscope autoradiography. Science, 163: 1203-1205.
Ney, R. L., N. J. Hochella, D. G. Grahame-Smith, R. N. Dexter and R. W. Butcher 1969 Abnormal regulation of
adenosine 3', 5'-monophosphate and corticosterone formation in an adrenocortical carcinoma. J. Clin. Invest.,
48: 1733-1739.
Nickerson, P. A. 1973 Adrenocortical cells in rats bearing a corticosterone secreting tumor. Virch. Arch. Abt. B
Zellpath., 13: 297-305.
Nielsen, E.. and V. H. Asfeldt 1967 Studies on the speci
ficity of fluorimetric determination of plasma corticosteroids ad Modurn de Moor and Steeno. Scand. J.
Clin. Lab. Invest., 20: 185-194.
Peron, F. G., A. Haksar, M. Lin, D. Kupfer, W. Robidoux, Jr.,
G. Kimmel and E. Bedigian 1974 Studies on respiration
and 1lp-hydroxylation of deoxycorticosterone in mitochondria and intact cells isolated from the Snell adrenocortical carcinoma 494. Cancer Res., 34: 2711-2719.
Schorr, I., and R. L. Ney 1971 Abnormal hormone responses
of an adrenocortical cancer adenyl cyclase. J. Clin.
Invest., 50: 1295-1300.
Schorr, I., B. Saxena and R. L. Ney 1971 Specificity of the
adenyl cyclase receptors of a n adrenocortical cancer. Endocrin. SOC.53rd Ann. Mtg., San Francisco, 90.
Sharma, R. K. 1973 Metabolic regulation of steroidogenesis in adrenocortical carcinoma cells of rat. Eur. J.
Biochem., 32: 506-512.
Sharma, R. K., and J. S. Brush 1973 Metabolic regulation
of steroidogenesis in isolated adrenal and adrenocortical
carcinoma cells of rat. The incorporation of (20Sb20(7-3H)hydroxycholesterolinto deoxycorticosterone and
corticosterone. Arch. Biochem. Biophys., 156: 560-562.
Sharma, R. K., and K. Hashimoto 1972 Ultrastructural
studies and metabolic regulation of isolated adrenocortical carcinoma cells of rat. Cancer Res., 32: 666-674.
Skipski, V. P., and M. Barclay 1969 Thin-layer chromatography of lipids. In: Methods in Enzymology. J . M. Lowenstein, ed. Academic Press, New York, pp. 530-598.
Snell, K. C., and H. L. Stewart 1959 Variations in histologic
pattern and functional effects of a transplantable adrenal
cortical carcinoma in intact, hypophysectomized, and
newborn rats. J. Nat'l. Cancer Inst., 22: 1119-1155.
Sperry, W. M.. and F. C. Brand 1955 The determination of
total lipids in blood serum. J. Biol. Chem., 213: 69-76.
Spurr, A. R. 1969 A low-viscosity epoxy resin embedding
medium for electron microscopy. J. Ultrastruct. Res., 26:
31-43.
Sweat, M. L., and M. J. Bryson 1964 Loss of two steroid 11
B-hydroxylating components in a r a t adrenocortical carcinoma. J. Nat'l. Cancer Inst., 33: 849-854.
Symington, T. 1969 Functional Pathology of the Human
Adrenal Gland. Baltimore: Williams and Wilkins.
Weibel, E. R. 1969 Stereological principles for morpbometry in electron microscopic cytology. Int. Rev. Cytol.,
26: 235-302.
PLATES
PLATE 1
EXPLANATION OF FIGURES
4 Adrenocortical tumor 494 cells, recovered seven days following detection of successful
transplantation, are characterized by profiles of ribosome-studded rough endoplasmic
reticulum (R), scattered cisternae of smooth endoplasmic reticulum ( S ) ,round to ovoid
mitochondria (MI, and a prominent Golgi complex (GI. In contrast to normal adrenocortical cells (fig. 6), some tumor cell mitochondria are elongated and others exhibit
poorly developed internae. Numerous free ribosomes (arrowheads) are found throughout the cytoplasm. X 20,400.
5 Portions of cells recovered from a 14-daytumor. Mitochondria have less electron-dense
matrices and few membranous internae. Note the close relationship of the stacked
tubules or plates of smooth endoplasmic reticulum to the mitochondria near the large
lipid droplet (L) (arrow). Coated vesicles can also be noted (arrowhead). X 13,900.
6
712
Normal adrenocortical zona fasciculata cells from a 7-day control rat. In contrast to
tumor cells, the smooth endoplasmic reticulum (arrowhead) is much more prominent
than rough. Mitochondria1 profiles are round and contain numerous vesicular internae. Lipid droplets (L) are considerably smaller in size and much more numerous than
those found in tumor cells. X 11,466.
STRUCTURE A N D FUNCTION OF RAT ADRENOCORTICAL TOMOH
Robert N Moore, David P Penney and Kathy A Averill
PLATE 1
713
PLATE 2
EXPLANATION OF FIGURES
714
7
Portions of several cells from a 21-day tumor. Note the degenerating characteristics of
the cell a t the right (e.g.,dilated nuclear envelope and cisternae of the endoplasmic reticulum, and the marked loss of intramitochondrial membranes). With increasing
time following transplantation, smooth endoplasmic reticulum is observed much more
frequently than rough. X 12,700.
8
Cells from a 21-day tumor. Note the polysomal configurations (arrow), the appearances of lysosomal dense bodies (arrowheads), and the numerous microvilli extending
into the intercellular spaces. X 10,200.
STRUCTURE A N D FUNCTION OF RAT ADRENOCORTICAL TUMOR
Robert N . Moore, David P. Penney and Kathy A. Averill
PLATE 2
715
PLATE 3
EXPLANATION OF FIGURES
9 Cells from a 28-day tumor. Mitochondria contain cristae which are primarily in a
tubular configuration. The large lipid droplet (L)is surrounded by a more extensive
development of the smooth endoplasmic reticulum. Dense lysosome-like bodies
(arrowheads), indicative of autophagocytosis, are frequently observed. X 20,000.
10 Portions of cells from a 35-day tumor. Older tumors are characterized by increased
numbers of dense bodies and occasionally exhibited direct cell-cell junctions (arrow).
X 11,350.
716
STRUCTURE AND FUNCTION OF RAT ADRENOCORTICAL TUMOR
Robert N Moore, David P Penney and Kathy A Averill
PI.ATE 3
717
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