Rat Adrenocortical Carcinoma 494: An Integrated Structural, Stereological, and Biochemical Analysis1
ROBERT N. MOORE,? DAVID P. PENNEY 3 AND KATHY A. AVERILL Departments of Anatomy and Dental Research, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642
ABSTRACT Snell adrenocortical tumor 494 was implanted into male Sprague-Dawley rats and recovered 7, 14, 21, 28 or 35 days following initial detec- tion 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 chemo- therapeutic agents on tumor cells. Tumor cells possessed oval or elongated mito- chondrial profiles with tubular cristae, one or two very large (> 5 um) lipid drop- lets, abundant ribosomes and coated vesicles, and sparse rough and smooth endo- plasmic reticulum. Stereologic evaluation revealed that tumor lipid volume was 41% and mitochondrial volume 29% that of the 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 cor- ticosterone levels in tumor-bearing rats were elevated 3-fold by 14 days after ini- tial detection. The adrenals of the tumor-bearing host exhibited marked involu- tion, the extent of which was directly related to tumor size.
The Snell transplantable rat adrenocortical carcinoma 494 (Snell and Stewart, ‘59) is an excellent model for the study of adrenocortical neoplasia in that it was of spontaneous origin, is relatively insensitive to ACTH (Kimmel et al., ‘74; Ney et al., ‘69), and is corticosterone- secreting (Sharma, ‘73; Sharma and Brush, ‘73). For these reasons, this tumor resembles many human adrenocortical tumors (Syming- ton, ‘69). Kimmel et al. (‘74) have described large clear vesicles and deficiencies in the number and internal structure of mitochon- dria in transplanted tumor cells grown sub- cutaneously for four to six weeks. Isolated tumor cell preparations exhibit sparse num- bers of lipid droplets and mitochondria, and moderately well developed smooth endoplas- mic reticulum and Golgi complexes (Sharma and Hashimoto, ‘72). When compared to the normal adrenal gland, this tumor has been shown to be 1-10% as efficient in corticoste- rone production (Ney et al., ‘69) and has de- creased 113-hydroxylase and 5a-reductase ac- tivities (Johnson et al., ‘61; Kimmel et al., ‘74; Maynard and Cameron, ‘72; Peron et al., ‘74;
Sweat and Bryson, ‘64). Energy (ATP) for rapid tumor cell proliferation may be in part provided by an a-glycerol phosphate shuttle which compensates for the markedly de- creased 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 that of the normal adrenal (Ney et al., ‘69), its stimu- lation by epinephrine, norepinephrine and TSH suggested the specificity of the mem- brane 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 the number and sur- face area of internal mitochondrial vesicles (Nickerson, ‘73). Mitochondria were elongated in shape rather than spherical and the volume
Received Oct. 22, ‘76. Accepted Sept. 29, ‘77.
’ Supported by U.S.P.H.S. Grants CA 11198 and DE-00003 and American Cancer Society Grant IN-18N.
2 Present address: School of Dentistry, University of California, Los Angeles, California 90024.
3 To whom reprint requests should be addressed.
of mitochondria per cell was reduced. A signif- icant decrease in the volume and surface area of smooth endoplasmic reticulum was also ob- served. These morphological manifestations of suppressed adrenocortical function were at- tributed to the elevated blood levels of cor- ticosteroids produced by the tumor which negatively feeds back on the hypothalam pophyseal axis and decreases ACTH release.
Although certain morphologic and biochem- ical aspects of this tumor have been previously reported, there has been no integrated study utilizing such parameters in a specific tem- poral sequence following transplantation. It will be the purpose of this investigation to ascertain on a quantitative basis the inter- relationships of fine structure and lipid bio- chemistry of adrenocortical carcinoma 494 cells during their establishment and growth following implantation. In addition to provid- ing further information to aid in the charac- terization of the tumor, these data will serve as a baseline to which further studies involv- ing the effects of clinically-utilized chemo- therapeutic agents on tumor growth and me- tabolism will be compared.
MATERIALS AND METHODS
Male albino Sprague-Dawley rats weighing 80-100 g at the start of the experimentation were housed in an air conditioned room and exposed to a 12:12 hour light:dark cycle (6:00 A.M. to 6:00 P.M.). Purina Laboratory Chow and fresh drinking water were supplied ad libitum. The carcinoma was obtained from tumor-bearing hosts maintained in the labora- tory. Pieces of viable tumor approximately 2 mm3 in size were transplanted subcutaneous- ly via trocar into the dorsolumbar region of ether-anesthetized rats. When the implant became palpable, usually 5-10 mm in cutane- ously-measured diameter by seven to ten days, the tumors were measured along their long and short axes with a caliper and their mean diameters calculated.
To assess the influences of the growing tumor on the host animal, daily body weights and tumor diameters (2 axes) were recorded. Animals (10-15 per group) were sacrificed by decapitation 7, 14, 21, 28 and 35 days after the tumors became palpable. Normal animals (5-6 per group) of equivalent age served as con- trols. At sacrifice (10:00-11:00 A.M. for each designated time period), the tumors and nor- mal adrenals were removed, measured, and
weighed. Specimens of the tumor and adrenals were taken for fine structural and biochem- ical analyses, and trunk blood was collected for plasma corticosterone determinations.
Electron microscopy
Pieces of non-necrotic viable tumor, and normal adrenal were fixed in formaldehyde- glutaraldehyde (Karnovsky, ‘65) for three hours, rinsed overnight in 0.1 M sodium cacodylate buffer (pH 7.2), post-fixed for one hour in 1% OsO4, 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 experimen- tal group and processed as noted above. Stereologic data was obtained by the differen- tial 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 at least 50 elec- tron micrographs were taken of the upper right hand corners of the grid openings at a constant magnification of 3,100 x. These plates were then printed to a standard mag- nification of 13,330 x. The nuclear fraction was obtained directly from differential point counting while the cytoplasmic volume frac- tion was determined by subtracting the sum of the extracellular space, nucleus, and necrotic tissue fractions from the total volume.
Biochemistry
Lipids were separated from tumor and adrenal samples by homogenization in meth- anol followed by subsequent extraction in chloroform: methanol (1:1) (Sperry and Brand, ‘55). Lipid fractionation was achieved by thin layer chromatography using silica gel plates (EM Laboratories) which were de- veloped in petroleum ether:diethyl ether:ace- tic acid (90:10:1) (Skipski and Barclay, ‘69). Concentrations of cholesterol and cholesterol esters were determined colorimetrically using the Liebermann-Burchard procedure employ- ing acetic anhydride, acetic acid and sulfuric acid (86:82:25) (Kabara, ‘62). The phosphate content of the lipid fraction was analyzed by the method of Lindberg and Ernstner (‘56).
400
CONTROL
.
BODY WEIGHT (grams) (00)
350
O TUMOR-BEARING
A TUMOR SIZE
I
₹
?
2
300
a
2
250
0
TUMOR
?
MEAN DIAMETER (mm)(4)
PALPABLE
9
4
40
200-
4
30
150
8
A
2
5
A
100
4
20
§
O
10
50
0
4
8
12
16
20
24
28
32
36
40
44
DAYS AFTER TRANSPLANTATION
60
50
TOTAL ADRENAL WEIGHT (mg)
40
30
20
10
0
0
5
10
15
20
25
30
35
TUMOR WEIGHT (gm)
Plasma free corticosterone was measured by the 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 ab- solute adrenal weight was inversely related to the weight of the tumor (fig. 2), with marked adrenal atrophy being present in animals with tumors that exceeded 4 g (> 25 mm mean diameter).
Tumor morphology
Tumors of all stages of growth were com- posed of cells in close approximation to sinus- oids. Giant multinucleated or polyploid cells, which are present in many carcinomas, were not observed. Cells, which morphologically ap- peared to be degenerative, were found occa- sionally interspersed among the viable cells. Degenerative cells were more frequently ob- served farther from the vessel lumen, espe- cially in tumors of larger diameters, suggest- ing an 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 the endoplasmic reticu- lum (figs. 5, 6). Cells exhibiting such signs of degeneration were considered to be at an in- termediate stage in the continuum of the de- generative 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 to viable tumor cells. The 7- day tumor will be described and age-related changes will be subsequently noted.
Nuclei were mostly round to ovoid, not indented, predominantly euchromatic and usually possessed one to two nucleoli. In con- trast to normal adrenocortical mitochondria of the zona fasciculata (fig. 6), which have a rounded profile and a vesicular internal struc- ture, tumor cell mitochondria were smaller, less numerous, and were frequently elongated or pleomorphic in shape (fig. 4). The sparse in- tramitochondrial membranes were predomi- nantly long and tubular (characteristic of cells of the adrenal zona glomerulosa) or occa- sionally 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 um 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 pat- terns or free in the cytoplasm (fig. 4). In marked contrast to normal adrenocortical cells (fig. 6), the smooth endoplasmic reticu- lum (SER) was poorly defined and was usually noted only in small, scattered areas of the cy-
toplasm (fig. 4). Distended cisternae of either the SER or RER were infrequently encoun- tered 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 ves- icles (fig. 5) and lysosomal dense bodies, bounded by a single limiting membrane, were noted (fig. 8), although their presence from cell to cell was variable. Microvilli were regu- larly 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 capil- lary endothelium was fenestrated, and the surrounding basement membrane was essen- tially intact. No evidence of viral particles was observed.
While cells with the above morphologic fea- tures were present for all time periods inves- tigated, with advancing age, most tumors showed a general trend toward an increase in both the size of the lipid droplets and their juxtapositioning to profiles of the smooth en- doplasmic reticulum (fig. 9). Although the en- doplasmic reticulum remained poorly devel- oped throughout all time periods, there ap- peared to be a gradual shift from the rough to the smooth variety with increasing post- transplantation time (fig. 9). Mitotic activity remained prominent, despite the considerable size of the tumor. Intercellular spaces were larger, and cells exhibiting degenerative char- acteristics were more widespread, again sug- gesting that the tumor was outgrowing its blood supply.
Stereology
In the normal adrenal cortex, necrotic tis- sue is rarely seen. The tumor frequently con- tained necrotic cells and thus had a greater percentage for all experimental periods (table 1). The mean volume fraction of tumor cell nu- clei from all age groups was 2.5 times greater (range: 2.3-2.8 x ) than adrenal nuclei. There- fore, since the total parenchymal cell volume of the tumor was similar to that of the adre- nal cortex, the cytoplasmic volume (cellular minus nuclear volumes) of the tumor was cor- respondingly less. The volume fractions of mi- tochondria and lipid of the tumor averaged 29% (range: 21-38%) and 41% (range: 30- 71%), respectively, those of the normal adre-
| Volume fraction of cells and organelles of normal rat adrenal cortex and adrenocortical tumor 494 (mean percentage ± S.E.) | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Experimental group | Extra cellular space and non-parenchymal cells | Necrotic cells and tissue | Cell 2 | Cytoplasm 3 | Nucleus | Mitochondria | Lipid | Lysosomal dense bodies | Secondary lysosomes (auto +/or heterophagosomes) |
| I. 7 days | |||||||||
| Adrenal cortex | 8.9±1.8 | 0 | 91.1±1.7 | 81.3±1.8 | 9.8±0.6 | 49.5±2.0 | 11.7±1.2 | 0.1±0.3 | 0 |
| Tumor 494 | 11.3± 1.5 | 8.3±1.8 | 80.4±1.7 | 55.4±1.6 | 25.0±2.1 | 10.5±0.6 | 3.6±1.0 | 0.6±0.1 | 0.6±0.2 |
| II. 14 days | |||||||||
| Adrenal cortex | 13.9±1.8 | 0 | 86.1±1.5 | 77.4±1.7 | 8.7±1.2 | 47.2±2.0 | 7.1± 1.0 | 0.8±0.2 | 0 |
| Tumor 494 | 8.6±0.7 | 1.3±0.6 | 90.1±1.3 | 65.7±2.1 | 24.4±1.4 | 13.5±0.6 | 2.5±0.5 | 0.6±0.1 | 0.4±0.1 |
| III. | |||||||||
| 21 days | |||||||||
| Adrenal cortex | 10.2±1.4 | 0.4±0.4 | 89.8± 1.4 | 78.0±1.5 | 11.4±1.4 | 50.6±2.2 | 5.1±0.6 | 0.8±0.2 | 0 |
| Tumor 494 | 7.5±1.0 | 8.7±2.3 | 83.8±1.6 | 57.6±1.8 | 26.2±2.3 | 12.6±0.6 | 3.6±1.0 | 0.7±0.1 | 0.3±0.1 |
| IV. | |||||||||
| 28 days | |||||||||
| Adrenal cortex | 8.0±1.3 | 0 | 92.0±1.2 | 82.1±1.3 | 9.9±1.1 | 42.4±1.8 | 16.5±1.9 | 0.6±0.1 | 0 |
| Tumor 494 | 17.2±1.1 | 1.2±0.7 | 81.6±1.6 | 57.8±1.5 | 23.8±1.7 | 13.7±0.6 | 4.9±1.0 | 0.3±0.1 | 0.1±0.1 |
| V. 35 days | |||||||||
| Adrenal cortex | 10.0±1.3 | 0 | 90.0±1.3 | 80.6+1.4 | 9.4±1.3 | 42.9±1.5 | 13.1±1.4 | 0.7±0.1 | 0 |
| Tumor 494 | 13.5±0.8 | 0.2±0.2 | 86.3±1.8 | 63.9±1.2 | 22.4±2.3 | 16.1±0.9 | 5.3±0.8 | 0.7±0.8 | 0.1±0.1 |
‘Days following initial tumor palpation or equivalent in normal.
2 Cell = Total volume - (extracellular and necrotic).
3 Cytoplasm = Cell - nucleus.
| Experimental group | Cholesterol (C) | Cholesterol esters (CE) | Ratio C/CE | Lipid phosphate |
|---|---|---|---|---|
| I. 7 days | ||||
| Adrenal | 1.74±0.032 | 13.59±0.25 | 0.13±0.01 | 0.22±0.01 |
| Tumor | 0.60±0.02 | 0.08±0.01 | 8.22±1.07 | 0.07±0.01 |
| II. 14 days | ||||
| Adrenal | 1.18±0.18 | 14.22±1.16 | 0.08±0.02 | 0.25±0.02 |
| Tumor | 0.73±0.04 | 0.18±0.02 | 4.63±0.54 | 0.07±0.01 |
| III. 21 days | ||||
| Adrenal | 1.48±0.13 | 16.93±2.28 | 0.09±0.01 | 0.29±0.03 |
| Tumor | 1.06±0.08 | 0.50± 0.04 | 2.21±0.26 | 0.08±0.01 |
| IV. 28 days | ||||
| Adrenal | 1.21±0.08 | 11.85±0.07 | 0.10±0.01 | 0.23±0.01 |
| Tumor | 0.58±0.04 | 0.32±0.03 | 1.88±0.15 | 0.06±0.01 |
| V. 35 days | ||||
| Adrenal | 1.33±0.24 | 14.40± 0.55 | 0.09±0.01 | 0.17±0.01 |
| Tumor | 0.79±0.06 | 0.53±0.06 | 1.59±0.16 | 0.03±0.01 |
1 Days following initial tumor palpation or equivalent in normal.
2 p < 0.001 for all values.
16
☒ NORMAL
PLASMA CORTICOSTERONE (ug/100ml)
14
☐ TUMOR-BEARING
12
10
8
6
4
2
7
14
21
28
35
EXPERIMENTAL PERIOD IN DAYS
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 progres- sive 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 re- mained 18-fold greater than for normal ad-
renal glands. Lipid phosphate was relatively consistent in all time groups and averaged 25% that of the normal adrenal. Although somewhat variable, plasma corticosterone levels of tumor-bearing rats were elevated, as much as 3-fold over normal control values (fig. 3), confirming the observations of Ney et al. (‘69).
DISCUSSION
The organellar fine structure of the car- cinoma cells exhibited considerable variabil- ity when compared to the organelles of the adrenocortices of normal animals. The much larger (2.5 times) nuclear volume fraction is consistent with the concept of a very rapidly metabolizing, aggressive neoplastic cell. The most striking deviations from normal occur in the cytoplasmic organelles, especially mito- chondria, lipid droplets, and endoplasmic re- ticulum. Similar observations have been re- ported in trypsinized tumor cells (Sharma and Hashimoto, ‘72) and in longer-term implants (Kimmel et al., ‘74).
When non-parenchymal cells, which are more prominent in the tumor, are deleted from consideration (table 1), there is no great dif- ference in the average amount of extracellu- lar space between the two tissues. Because of the tumor’s tendency to outgrow its blood sup- ply, there are many more necrotic cells than in the normal adrenal (table 1).
Kimmel et al. (‘74) have reported that on a protein/tissue net weight basis, there is a 75% reduction of mitochondria in tumor cells. The present stereologic data (table 1) are in close agreement and show a 71% reduction in the mitochondrial volume fraction.
The diminished numbers of mitochondria and their loss or reduction of internal mem- branes have been directly correlated with the concomitant reduction of mitochondrial 113- hydroxylase activity in tumor cells (Kimmel et al., ‘74). The deficiency of this enzyme, which in the rat controls the conversion of 11- deoxycorticosterone (DOC) to corticosterone, the principal glucocorticoid synthesized, con- tributes significantly to the overall inefficien- cy (< 10% of normal adrenal cortical cells) of the tumor cells in hormone biosynthesis and secretion (Ney et al., ‘69). The marked loss of mitochondrial matricial density and the pres- ence of intramitochondrial myelinated bodies (Kimmel et al., ‘74; Sharma and Hashimoto, ‘72) were observed in our studies only in those tumor cells which also exhibited other signs of
degradation (e.g., nucleolemma distention, RER and SER dilation) and were therefore considered to represent degenerated or degen- erating cells. As noted earlier, tumor cells have been shown to be ACTH-insensitive (Ney et al., ‘69). Since Kahri et al. (‘70) have shown the maintenance of the vesicular interna of mitochondria of the zona fasciculata to be dependent on available ACTH, the refractility of tumor cells to ACTH may be directly re- sponsible for the lamellar configuration of the intramitochondrial membranes.
The marked deviation of tumor cells from normal adrenocortical morphology is in the drastic reduction in number and increase in size of lipid dropets. Such changes are usually reflective of decreased hormone biosynthesis (Greep and Deane, ‘49). In normal steroid-se- creting tissues, these lipid droplets are reser- voirs of hormone precursors, cholesterol, and especially cholesterol esters (Moses et al., ‘69). However, in the adrenocortical tumor cells (table 2), the opposite is true and lipid, which is reduced to 41% that of normal adreno- cortices, is primarily cholesterol, and the ca- pacity to esterify cholesterol appears to be markedly reduced. Since similar findings were reported by Lossow et al. (‘65) for the 494H tumor line, a sub-line of the same tumor estab- lished by several passages through hypophy- sectomized hosts, it appears that the limited capacity to esterify cholesterol may be in- herent in the tumor line and may not be at- tributable solely to a functional alteration induced by transplantation into hormonally- deprived, hypophysectomized animals (494H).
With time, there is a progressive decrease in the cholesterol/cholesterol ester ratios in tu- mors (table 2), suggestive of an increasing ca- pacity of cells of more mature and established tumors to esterify cholesterol. This phenome- non appears to be directly correlated with the gradual increase in smooth endoplasmic retic- ulum (SER) of tumor cells with time (fig. 7). Since it has been proposed that the enzymes involved in cholesterol esterification occur in the SER, which is never as well developed in tumor cells as in normal adrenocortical cells, the efficiency of the tumor in cholesterol esterification will most likely remain signifi- cantly diminished even in longer periods of time post-transplantation.
Lipid phosphate of the tumor was about 25% that of the normal adrenal cortex. Assuming this parameter is predominantly an index of cytomembranes, the results were in agree-
ment with the fine structural data which revealed a marked decrease of the ER, both rough and smooth and mitochondrial internal membranes in tumor cells is compared to nor- mal adrenocortical cells.
Generally, the plasma corticosterone values for tumor-bearing rats (fig. 3) were approx- imately two to three times greater than con- trols for the first 21 days following establish- ment. After this time there was a decrease in the values almost to control levels, despite tumor enlargement (fig. 1), with presumably a concomitant increase in hormone production. This change most likely reflects the marked adrenal atrophy which is evident in tumor- bearing animals by this time (fig. 2). The corticosterone secreted by the tumor is efficacious in reducing ACTH secretion via the hypothalamushyseal-adrenocortical axis, and the adrenocortices, therefore, resem- ble 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 current- ly 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 sup- plying the adrenocortical carcinoma, to Dr. Guido V. Marinetti, University of Rochester, for assistance with the biochemical determi- nations, and to Mrs. Susan Walker for section- ing the tissue for electron microscopy.
LITERATURE CITED
Greep, R. O., and H. W. Deane 1949 Histological, cyto- chemical and physiological observations on the regenera- tion of the rat’s adrenal gland following enucleation. En- docrin., 45: 42-56.
Johnson, D. F., K. C. Snell, D. Francois and E. Heftmann 1961 In Vitro metabolism of progesterone-4- 4℃ 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 chloram- phenicol of ACTH-induced reorganization of inner mito- chondrial membranes in fetal adrenal cortical cells in tis- sue 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, Jr. 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 a transplantable adrenal cortical tumor. Proc. Soc. Exp. Biol. Med., 119: 125-131.
Maynard, P. V., and E. H. D. Cameron 1972 Metabolism of C19-steroids by homogenates of normal rat and mouse adrenal tissue and of the Snell transplantable rat adreno- cortical 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 micro- scope autoradiography. Science, 163: 1203-1205.
Ney, R. L., N. J. Hochella, D. G. Grahame-Smith, R. N. Dex- ter and R. W. Butcher 1969 Abnormal regulation of adenosine 3’, 5’-monophosphate and corticosterone for- mation in an adrenocortical carcinoma. J. Clin. Invest., 48: 1733-1739.
Nickerson, P. A. 1973 Adrenocortical cells in rats bear- ing a corticosterone secreting tumor. Virch. Arch. Abt. B Zellpath., 13: 297-305.
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Peron, F. G., A. Haksar, M. Lin, D. Kupfer, W. Robidoux, Jr., G. Kimmel and E. Bedigian 1974 Studies on respiration and 110-hydroxylation of deoxycorticosterone in mito- chondria and intact cells isolated from the Snell adreno- cortical 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 an adrenocortical cancer. En- docrin. Soc. 53rd Ann. Mtg., San Francisco, 90.
Sharma, R. K. 1973 Metabolic regulation of steroido- genesis 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 (20S)-20- (7-3H)hydroxycholesterol into deoxycorticosterone and corticosterone. Arch. Biochem. Biophys., 156: 560-562.
Sharma, R. K., and K. Hashimoto 1972 Ultrastructural studies and metabolic regulation of isolated adrenocorti- cal carcinoma cells of rat. Cancer Res., 32: 666-674.
Skipski, V. P., and M. Barclay 1969 Thin-layer chromatog- raphy of lipids. In: Methods in Enzymology. J. M. Lowen- stein, 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.
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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 (M), and a prominent Golgi complex (G). In contrast to normal adreno- cortical cells (fig. 6), some tumor cell mitochondria are elongated and others exhibit poorly developed internae. Numerous free ribosomes (arrowheads) are found through- out the cytoplasm. x 20,400.
5 Portions of cells recovered from a 14-day tumor. 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 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. Mitochondrial profiles are round and contain numerous vesicular inter- nae. Lipid droplets (L) are considerably smaller in size and much more numerous than those found in tumor cells. x 11,466.
STRUCTURE AND FUNCTION OF RAT ADRENOCORTICAL TUMOR Robert N. Moore, David P. Penney and Kathy A. Averill
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PLATE 2 EXPLANATION OF FIGURES
7 Portions of several cells from a 21-day tumor. Note the degenerating characteristics of the cell at the right (e.g., dilated nuclear envelope and cisternae of the endoplasmic re- ticulum, and the marked loss of intramitochondrial membranes). With increasing time following transplantation, smooth endoplasmic reticulum is observed much more frequently than rough. × 12,700.
8 Cells from a 21-day tumor. Note the polysomal configurations (arrow), the appear- ances of lysosomal dense bodies (arrowheads), and the numerous microvilli extending into the intercellular spaces. x 10,200.
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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). × 11,350. ☒
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