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Cholesterol Accumulation in Heterotransplanted Renal Cell Cancer
RALPH V. CLA From the Departments
"RICARDO
ARTHUR Y. ELLIOTT,t DONALD E. GLEASON MARY E. DEMPSEY
AND
Urologic Surgery and Biochemistry, University of IVlinnesota College of Health Sciences and the Department of Pathology, Fairview Hospital, Minneapolis, Minnesota
ABSTRACT
rro date there have been no in vivo studies of the knovvn ~··~~~._:r~,,,";",:-,.· of human renal cell cancer to accumulate amounts of cholesterol. a .. -,.. ,~" RCC line, was grown in the cheek of""·'"~"'~~ nnr"'"""'rl Sy:r.ian hamsters. of cholesterol levels revealed 2 to 4 times as in the heterotransplants as in any of the host tissues. After of tumor-bearing hamsters with 4- 14 C-cholesterol, the tumors accumulated than did hamster liver and no more radioactivity than did hamster or cheek It appears that accumulation of cholesterol by human RCC is not dependent on extensive of extracellular cholesterol. Instead, an intrinsic defect in the cellular regulation of cholesterol ,,,,v.,-·"·~ and efflux is more to be responsible. Cholesterol accumulation a hallmark of most renal cell 1• 2 and thefr abundant content of this substance cancers accounts in part for their clear-cell appearance in conventional ntst
,w,m. ,u'r.,
of appears to the origin of mechanism
v,•vv.,,v,u,vcu
mccn,,wcc,,cu
male 4 mg. of cortisone acetate
w1ca11uu"'
h~,,~~-"
hamsters host organs studied. up amounts of cholesterol, and it appears that human RCC results from a mechanism other
NIATERIALG AND METHODS
Cell cultures
cell line established from a skin Dr. of the Research, N' evv York. 11,l e havs other studies of
0.3 units of U.S.P. V.fhen the cells had formed a '"'°'"·Jw,., were subcultured with 0.25 pe:r cent (Difeo) and 1:5000 Vernene 0.02 per cent minetet:raacetic acid; Gene:ral Ohio). Cells from the 1st QHQ<'>Dn,ri
hamsters were anesthetized with cortisone acetate. The cheek pv~,.. ,,,v were rn,~,,u.n 100 of either culture medium or killed-cell C, 30 minutes). l X 107 cells were into Hamsters were injected subcutaneously on days 9, 11 and 13 with 4 mg., 2 mg. and 2 mg. of cortisone acetate, 13 all hamsters 4- 14 C-cholesterol .1.Ai:,1.1JH, Massachusetts) in 0.99 ml. of normal saline 0.01 ml. of absolute ethanol. Pairs of hamsters were killed 7, 24, 48 and 99 hours The tumors, left cheek µu,uc.u,s.s, right of liver were removed, blotted to remove ,,.c,.,,,,cu to the nearest 0.1 mg. in tightly capped tubes. methods. The tissue samples were minced and l ml. of 15 per cent ethanol vvas added for each 100 mg. in a lOOC water bath for 2 and their
were
an eEor of 1 per cent. RESULTS
Tumor mr·rrn·urnn Tumors grew in all hamsters injected with live Caki-1 cells. By 13 (8 days after "'"'"n,,,n of cells) the well-vascularized lesions invaded the cheek pouch epithelium (fig. 1). They were devoid of round-cell infiltrates. The cytoplasm of the tumor cells appeared less granular than that of Caki-1 cells in vitro (fig. 2). Karyotyping revealed both human and hamster chromosomes in the tumors as other investigators have reported. 7 No tumors were seen in the cheek pouches injected with culture medium or heat-killed cells. Cholesterol content. The cheek pouch RCC took up less 414C-cholesterol than did liver. However, its rate of uptake was
622
CLAYMAN AND ASSOCIATES
96 . - - - - - - - - - - - - - - - - - - - - - - - - , o Tumor
90
• Kidney
84
o Liver • Cheek pouch
78 72
66
:::J
(/) (/)
60 CJl
E
54
'E
0. "'O
48
-
42
0
36
(/)
C :::J
u
30 24 18
l2
:~~ FIG. 1. Cheek pouch tumors. A, 2nd day; although most of the cells in the center appear to be degenerating there is an area of early tumor formation along the cheek pouch epithelium. H & E, reduced from X65. B, 6th day; the well-established tumor has many vascular channels and is invading the cheek pouch epithelium. H & E, reduced from X65.
0 4
24
100
48
Time ( hours) FIG. 3. Total radioactivity per milligram of tissue specimen of heterotransplanted Caki-1 cheek pouch tumor, hamster kidney, liver and contralateral (tumor-free) cheek pouch.
14 O Tumor
12
-C
Q) +-
en en
C
:::J
O·u-
.. E
-
e'
en .c: Q)
0
.c
10
8
6
(.)
0)
E
u-
similar to that of normal cheek pouch, and, at later time points, to normal kidney (fig. 3). The cholesterol content of the tumors was consistently 2- to 4-fold greater than that of any of the other tissues examined (fig. 4). Except in specimens analyzed 7 hours after injection the tumors maintained a significantly lower specific activity than did any of the other tissues (fig. 5).
D liver
• Che81rpouch
0)
Q).....: +- 0
FIG. 2. A, Caki-1 cell line in vitro. Phase contrast, reduced from X 300. B, cheek pouch Caki-1 tumor on 6th day in vivo. H & E, reduced from X 400.
• Kidney
4
2 0
0
20
40
60
80
100
Time (hours) FIG. 4. Total milligrams of cholesterol per gm. of wet weight of tissue from heterotransplanted Caki-1 tumor, hamster kidney, liver and contralateral (tumor-free) cheek pouch.
623
nee
-r~--
?1 . 000 1
o
's,ooc -· L I 15,000 -
13,000
~
r
i------
.----4
liJmcr
iJj;
D Liver
1
E 9,ooo Q
u
6,000
3,000
FIG. 5, of normal hamster tissues compared with that of hetei:otraiuS!)lant,ea Caki-1 cheek pouch tumors. DISCUSSION
The aim of our was to determine whether the cholesterol in human RCC is of intracellular or extracellular To ~~~~~~~"'~'" this, we induced solid Caki-1 tumors in the cheek po,uc:nE,s unc,si:1pJJn1ss:ea hamsters; the absence of a CH'""'~"··abundant connective tissue and the "'"'""'~"'"'""":, of pouch make it favorable and useful for heterotransplantation of human cancers. 8 - 10 The results obtained with this model a:re valid that all studies are completed ,Nithin 3 weeks of tumor inoculation. 9 · 11 Our rate of tumor induction with Caki-1 cells v,as 100 per cent and all data were coHected within 12 of inocul&.tion. In order to obtain successful tumor ehmmi"fft, it was necessarv to inject high doses of cortisone acetate. The effect of corticost~roid on cholesterol metabolism to be minimal. Studies in rats have revealed that increase the of the rate cholesterol m·ociuc,n,Jn. increase in cholesterol Furthermore, the administration of steroids has never been shown to lead to the storage of cholesterol either in ,u,c>n;;uau tissues. 12 Hence, we believe that the storage of ~vv"0~,c~i seen in RCC in our m un:related to steroid administration. Earher in vivo studies of tu::-r10r cholesterol metabolism were done with :rat and human ne,pE,tcm-:as. WeH,,differentiated atGmas IT.tanifest norrnal vvhereas differentiated do not tumors neither he.ve a clear·~cell appearance nor acc:J.mulate cholesterol ,, o.
LHoon,~«~,
A feattr:e of the in. vitro n1odel for RCC is that the cultu:ced cancer cells lose their cles.r cell appearance, Indeed, the cells have 2. ~,m,u,,-, appearance and often contain less cholesternl than do benign cells under culture ~v·u,Ho,~,.,u. 5 " 15 The reason for the difference between the appearance of the RCC cell in vitro and in vivo may relate to the physical environment. In vitro the vast overlying pool of rnedium may enhance the release of cholesterol, whereas in vivo the of extracellular fluid available to receive the sterol would be considerably less per cell. Alternatively, the content of high density lipoproteins and other substances capable of leaching cholesterol from the cell may be higher in the culture medium than in the extracellular fluid present in vivo. . The results of the present in vivo studies of cholesterol are in agreement with ou:r earlier findings in vitro in that the RCC cells absorbed no more, and in some cases less, radioactive cholesterol than did normal kidney cells or any of the other benign tissues studied, However, the cheek pouch tumors resembled the dear-cell tumors histologically, and routinely contained 2 to 4 times as much cholesterol as did any of the host tissues, Accordingly, the accumulation of cholesterol by RCC in this in vivo model must result from some process other than uptake of extracellular steroL In our view, the cholesterol probably is of intracellular origin and the result of any of 3 mechanisms, alone or in combination. First, the membrane of the RCC cell may lack the surface receptors for absorbing lowor other regulatory substances. As a in,,,.nPnnr~t 0 the materials able to control intracellular sterol ou-.-.rnc,Q,o found in cells from µo,""'"u"~ with familial hypercholesterolemia.17 Second, the problem may be an aberration in cholesterol esterification; cells rich in free cholesterol often it for 18 Interestingly, cholesterol in the RCC cell is predomin the esterified state, whereas most of the cholesterol in the normal kidney cell is unesterified. 19 Esters could accumulate either because of increased of the enzyme re:,p(m1,1ble for the reaction, cholesterol transferase, or because of decreased of esters already formed. Aberrations in cholesterol esterification have been identified in 2 human u~nuvo·~o. 1Nolman's disease and cholesteryl ester disease. 20 the membrane of the RCC cell in vivo unable to release cholesterol effectively, thereby leading inside the cell. N ormaHy, a lipoprotein (HDL), access of HDL (or substances) to the cell surface, or membrane with regard to cholesterol. efr'1ux, could cause the buildup of intracellular ste:roL Studies with ott'.er types of cells have corroborated the importance of these '-,U•uico•a0, _-c,m,",,m,,c; substances; the addition of HDL to tissue culture media decreases the cholesterol ester content of mt aortic smooth muscle eel.ls and it has been that HDL has a similar function in human ceHs. 21 Further studies of cholesterol sy:rn1r1e:,rn and regulation, esterification and efflux are needed to better elucidate the biochemical to cholesterol accumulation in renal cancer. An n,,,a1.,u1ub of metabolism in clear-cell tumors may permit more effective u.«,;;1m~"'" and therapeutic approaches to this cancer. REFERENCES L Leary, T.: Crystalline ester cholesterol and adult renal tumors. Arch. PathoL, 50: 151, 1950, 2. Lindlar, F.: Hypernephroides Karzinom und Nierenkarzinom: Lipidchemische Analyse von 24 Nierentumoren, Verh. Dtsch, Ges, Pathol., 45: 144, 1961. 3. Bennington, J. L.: Histopathology of renal adenocarcinoma. Un.
624
CLAYMAN AND ASSOCIATES
Int. Cancer Tech. Rep. Ser., 49: 61, 1980. 4. Fogh, J. and Trempe, G.: New human tumor cell lines. In: Human Tumor Cells In Vitro. Edited by J. Fogh. New York: Plenum Press, p. 115, 1975. 5. Gonzalez, R., Dempsey, M. E., Elliott, A. Y. and Fraley, E. E.: Studies of sterol synthesis, synthesis regulation and transport in cultured human kidney. Exp. Cell Res., 87: 152, 1974. 6. Abell, L. L., Levy, B. B., Brodie, B. D. and Kendell, F. E.: A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J. Biol. Chem., 195: 357, 1952. 7. Goldenberg, D. M., Pavia, R. A. and Tsao, M. C.: In vivo hybridisation of human tumour and normal hamster cells. Nature, 250: 649, 1974. 8. Foley, G. E. and Handler, A. H.: Differentiation of "normal" and neoplastic cells maintained in tissue culture by implantation into normal hamsters. Proc. Soc. Exp. Biol. Med., 94: 661, 1957. 9. Patterson, W. B.: Transplantation of human cancers to hamster cheek pouches. Cancer Res., 28: 1637, 1968. 10. Patterson, W. B., Chute, R. N. and Sommers, S. C.: Transplantation of human tumors into cortisone-treated hamsters. Cancer Res., 14: 656, 1954. 11. Fogh, J.: Reduced tumor-producing capacity of mycoplasma-modified lines of FL human amnion cells in the cheek pouch of cortisonized hamsters. Cancer Res., 29: 1721, 1969. 12. Mitropoulos, K. A. and Balasubramaniam, S.: The role of glucocorticoids in the regulation of the diurnal rhythm of hepatic /3hydroxy-/3-methylglutaryl-coenzyme A reductase and cholesterol 7a-hydroxylase. Biochem. J., 160: 49, 1976.
13. Mitchell, A. D., Pugh, T. D. and Goldfarb, S.: Partial "feedback control" of /3-hydroxy-/3-methylglutaryl coenzyme A reductase activity in primary hepatocellular carcinomas. Cancer Res., 38: 4474, 1978. 14. Siperstein, M. D., Fagan, V. M. and Morris, H.P.: Further studies on the deletion of the cholesterol feedback system in hepatomas. Cancer Res., 26: 7, 1966. 15. Gonzalez, R. and Dempsey, M. E.: Sterol synthesis in cultured human renal cell cancer. J. Urol., 117: 708, 1977. 16. Clayman, R. V., Gonzalez, R., Elliott, A. Y. and Dempsey, M. E.: Differences in cholesterol transport by normal and malignant human kidney cells in culture. Biochem. Biophys. Res. Comm., 92: 1355, 1980. 17. Brown, M. D., Kovanen, P. T. and Goldstein, J. L.: Regulation of plasma cholesterol by lipoprotein receptors. Science, 212: 628, 1981. 18. Brown, M. S., Dana, S. E. and Goldstein, J. L.: Cholesterol ester formation in cultured human fibroblasts. J. Biol. Chem., 250: 4025, 1975. 19. Gonzalez, R., Clayman, R. V. and Dempsey, M. E.: Cholesterol accumulation in renal cancer-a review. Invest. Urol., 19: 1, 1981. 20. Sloan, H. R. and Fredrickson, D. S.: Rare familial diseases with neutral lipid storage. In: The Metabolic Basis of Inherited Disease. Edited by J. B. Stanbury, J. B. Wyngaarden and D. S. Fredrickson. Ed. 3. New York: McGraw Hill, p. 808, 1972. 21. Stein, 0., VanderHoek, J. and Stein, Y.: Cholesterol ester accumulation in cultured aortic smooth muscle cell. Atherosclerosis, 26: 465, 1977.
"RICARDO
ARTHUR Y. ELLIOTT,t DONALD E. GLEASON MARY E. DEMPSEY
AND
Urologic Surgery and Biochemistry, University of IVlinnesota College of Health Sciences and the Department of Pathology, Fairview Hospital, Minneapolis, Minnesota
ABSTRACT
rro date there have been no in vivo studies of the knovvn ~··~~~._:r~,,,";",:-,.· of human renal cell cancer to accumulate amounts of cholesterol. a .. -,.. ,~" RCC line, was grown in the cheek of""·'"~"'~~ nnr"'"""'rl Sy:r.ian hamsters. of cholesterol levels revealed 2 to 4 times as in the heterotransplants as in any of the host tissues. After of tumor-bearing hamsters with 4- 14 C-cholesterol, the tumors accumulated than did hamster liver and no more radioactivity than did hamster or cheek It appears that accumulation of cholesterol by human RCC is not dependent on extensive of extracellular cholesterol. Instead, an intrinsic defect in the cellular regulation of cholesterol ,,,,v.,-·"·~ and efflux is more to be responsible. Cholesterol accumulation a hallmark of most renal cell 1• 2 and thefr abundant content of this substance cancers accounts in part for their clear-cell appearance in conventional ntst
,w,m. ,u'r.,
of appears to the origin of mechanism
v,•vv.,,v,u,vcu
mccn,,wcc,,cu
male 4 mg. of cortisone acetate
w1ca11uu"'
h~,,~~-"
hamsters host organs studied. up amounts of cholesterol, and it appears that human RCC results from a mechanism other
NIATERIALG AND METHODS
Cell cultures
cell line established from a skin Dr. of the Research, N' evv York. 11,l e havs other studies of
0.3 units of U.S.P. V.fhen the cells had formed a '"'°'"·Jw,., were subcultured with 0.25 pe:r cent (Difeo) and 1:5000 Vernene 0.02 per cent minetet:raacetic acid; Gene:ral Ohio). Cells from the 1st QHQ<'>Dn,ri
hamsters were anesthetized with cortisone acetate. The cheek pv~,.. ,,,v were rn,~,,u.n 100 of either culture medium or killed-cell C, 30 minutes). l X 107 cells were into Hamsters were injected subcutaneously on days 9, 11 and 13 with 4 mg., 2 mg. and 2 mg. of cortisone acetate, 13 all hamsters 4- 14 C-cholesterol .1.Ai:,1.1JH, Massachusetts) in 0.99 ml. of normal saline 0.01 ml. of absolute ethanol. Pairs of hamsters were killed 7, 24, 48 and 99 hours The tumors, left cheek µu,uc.u,s.s, right of liver were removed, blotted to remove ,,.c,.,,,,cu to the nearest 0.1 mg. in tightly capped tubes. methods. The tissue samples were minced and l ml. of 15 per cent ethanol vvas added for each 100 mg. in a lOOC water bath for 2 and their
were
an eEor of 1 per cent. RESULTS
Tumor mr·rrn·urnn Tumors grew in all hamsters injected with live Caki-1 cells. By 13 (8 days after "'"'"n,,,n of cells) the well-vascularized lesions invaded the cheek pouch epithelium (fig. 1). They were devoid of round-cell infiltrates. The cytoplasm of the tumor cells appeared less granular than that of Caki-1 cells in vitro (fig. 2). Karyotyping revealed both human and hamster chromosomes in the tumors as other investigators have reported. 7 No tumors were seen in the cheek pouches injected with culture medium or heat-killed cells. Cholesterol content. The cheek pouch RCC took up less 414C-cholesterol than did liver. However, its rate of uptake was
622
CLAYMAN AND ASSOCIATES
96 . - - - - - - - - - - - - - - - - - - - - - - - - , o Tumor
90
• Kidney
84
o Liver • Cheek pouch
78 72
66
:::J
(/) (/)
60 CJl
E
54
'E
0. "'O
48
-
42
0
36
(/)
C :::J
u
30 24 18
l2
:~~ FIG. 1. Cheek pouch tumors. A, 2nd day; although most of the cells in the center appear to be degenerating there is an area of early tumor formation along the cheek pouch epithelium. H & E, reduced from X65. B, 6th day; the well-established tumor has many vascular channels and is invading the cheek pouch epithelium. H & E, reduced from X65.
0 4
24
100
48
Time ( hours) FIG. 3. Total radioactivity per milligram of tissue specimen of heterotransplanted Caki-1 cheek pouch tumor, hamster kidney, liver and contralateral (tumor-free) cheek pouch.
14 O Tumor
12
-C
Q) +-
en en
C
:::J
O·u-
.. E
-
e'
en .c: Q)
0
.c
10
8
6
(.)
0)
E
u-
similar to that of normal cheek pouch, and, at later time points, to normal kidney (fig. 3). The cholesterol content of the tumors was consistently 2- to 4-fold greater than that of any of the other tissues examined (fig. 4). Except in specimens analyzed 7 hours after injection the tumors maintained a significantly lower specific activity than did any of the other tissues (fig. 5).
D liver
• Che81rpouch
0)
Q).....: +- 0
FIG. 2. A, Caki-1 cell line in vitro. Phase contrast, reduced from X 300. B, cheek pouch Caki-1 tumor on 6th day in vivo. H & E, reduced from X 400.
• Kidney
4
2 0
0
20
40
60
80
100
Time (hours) FIG. 4. Total milligrams of cholesterol per gm. of wet weight of tissue from heterotransplanted Caki-1 tumor, hamster kidney, liver and contralateral (tumor-free) cheek pouch.
623
nee
-r~--
?1 . 000 1
o
's,ooc -· L I 15,000 -
13,000
~
r
i------
.----4
liJmcr
iJj;
D Liver
1
E 9,ooo Q
u
6,000
3,000
FIG. 5, of normal hamster tissues compared with that of hetei:otraiuS!)lant,ea Caki-1 cheek pouch tumors. DISCUSSION
The aim of our was to determine whether the cholesterol in human RCC is of intracellular or extracellular To ~~~~~~~"'~'" this, we induced solid Caki-1 tumors in the cheek po,uc:nE,s unc,si:1pJJn1ss:ea hamsters; the absence of a CH'""'~"··abundant connective tissue and the "'"'""'~"'"'""":, of pouch make it favorable and useful for heterotransplantation of human cancers. 8 - 10 The results obtained with this model a:re valid that all studies are completed ,Nithin 3 weeks of tumor inoculation. 9 · 11 Our rate of tumor induction with Caki-1 cells v,as 100 per cent and all data were coHected within 12 of inocul&.tion. In order to obtain successful tumor ehmmi"fft, it was necessarv to inject high doses of cortisone acetate. The effect of corticost~roid on cholesterol metabolism to be minimal. Studies in rats have revealed that increase the of the rate cholesterol m·ociuc,n,Jn. increase in cholesterol Furthermore, the administration of steroids has never been shown to lead to the storage of cholesterol either in ,u,c>n;;uau tissues. 12 Hence, we believe that the storage of ~vv"0~,c~i seen in RCC in our m un:related to steroid administration. Earher in vivo studies of tu::-r10r cholesterol metabolism were done with :rat and human ne,pE,tcm-:as. WeH,,differentiated atGmas IT.tanifest norrnal vvhereas differentiated do not tumors neither he.ve a clear·~cell appearance nor acc:J.mulate cholesterol ,, o.
LHoon,~«~,
A feattr:e of the in. vitro n1odel for RCC is that the cultu:ced cancer cells lose their cles.r cell appearance, Indeed, the cells have 2. ~,m,u,,-, appearance and often contain less cholesternl than do benign cells under culture ~v·u,Ho,~,.,u. 5 " 15 The reason for the difference between the appearance of the RCC cell in vitro and in vivo may relate to the physical environment. In vitro the vast overlying pool of rnedium may enhance the release of cholesterol, whereas in vivo the of extracellular fluid available to receive the sterol would be considerably less per cell. Alternatively, the content of high density lipoproteins and other substances capable of leaching cholesterol from the cell may be higher in the culture medium than in the extracellular fluid present in vivo. . The results of the present in vivo studies of cholesterol are in agreement with ou:r earlier findings in vitro in that the RCC cells absorbed no more, and in some cases less, radioactive cholesterol than did normal kidney cells or any of the other benign tissues studied, However, the cheek pouch tumors resembled the dear-cell tumors histologically, and routinely contained 2 to 4 times as much cholesterol as did any of the host tissues, Accordingly, the accumulation of cholesterol by RCC in this in vivo model must result from some process other than uptake of extracellular steroL In our view, the cholesterol probably is of intracellular origin and the result of any of 3 mechanisms, alone or in combination. First, the membrane of the RCC cell may lack the surface receptors for absorbing lowor other regulatory substances. As a in,,,.nPnnr~t 0 the materials able to control intracellular sterol ou-.-.rnc,Q,o found in cells from µo,""'"u"~ with familial hypercholesterolemia.17 Second, the problem may be an aberration in cholesterol esterification; cells rich in free cholesterol often it for 18 Interestingly, cholesterol in the RCC cell is predomin the esterified state, whereas most of the cholesterol in the normal kidney cell is unesterified. 19 Esters could accumulate either because of increased of the enzyme re:,p(m1,1ble for the reaction, cholesterol transferase, or because of decreased of esters already formed. Aberrations in cholesterol esterification have been identified in 2 human u~nuvo·~o. 1Nolman's disease and cholesteryl ester disease. 20 the membrane of the RCC cell in vivo unable to release cholesterol effectively, thereby leading inside the cell. N ormaHy, a lipoprotein (HDL), access of HDL (or substances) to the cell surface, or membrane with regard to cholesterol. efr'1ux, could cause the buildup of intracellular ste:roL Studies with ott'.er types of cells have corroborated the importance of these '-,U•uico•a0, _-c,m,",,m,,c; substances; the addition of HDL to tissue culture media decreases the cholesterol ester content of mt aortic smooth muscle eel.ls and it has been that HDL has a similar function in human ceHs. 21 Further studies of cholesterol sy:rn1r1e:,rn and regulation, esterification and efflux are needed to better elucidate the biochemical to cholesterol accumulation in renal cancer. An n,,,a1.,u1ub of metabolism in clear-cell tumors may permit more effective u.«,;;1m~"'" and therapeutic approaches to this cancer. REFERENCES L Leary, T.: Crystalline ester cholesterol and adult renal tumors. Arch. PathoL, 50: 151, 1950, 2. Lindlar, F.: Hypernephroides Karzinom und Nierenkarzinom: Lipidchemische Analyse von 24 Nierentumoren, Verh. Dtsch, Ges, Pathol., 45: 144, 1961. 3. Bennington, J. L.: Histopathology of renal adenocarcinoma. Un.
624
CLAYMAN AND ASSOCIATES
Int. Cancer Tech. Rep. Ser., 49: 61, 1980. 4. Fogh, J. and Trempe, G.: New human tumor cell lines. In: Human Tumor Cells In Vitro. Edited by J. Fogh. New York: Plenum Press, p. 115, 1975. 5. Gonzalez, R., Dempsey, M. E., Elliott, A. Y. and Fraley, E. E.: Studies of sterol synthesis, synthesis regulation and transport in cultured human kidney. Exp. Cell Res., 87: 152, 1974. 6. Abell, L. L., Levy, B. B., Brodie, B. D. and Kendell, F. E.: A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J. Biol. Chem., 195: 357, 1952. 7. Goldenberg, D. M., Pavia, R. A. and Tsao, M. C.: In vivo hybridisation of human tumour and normal hamster cells. Nature, 250: 649, 1974. 8. Foley, G. E. and Handler, A. H.: Differentiation of "normal" and neoplastic cells maintained in tissue culture by implantation into normal hamsters. Proc. Soc. Exp. Biol. Med., 94: 661, 1957. 9. Patterson, W. B.: Transplantation of human cancers to hamster cheek pouches. Cancer Res., 28: 1637, 1968. 10. Patterson, W. B., Chute, R. N. and Sommers, S. C.: Transplantation of human tumors into cortisone-treated hamsters. Cancer Res., 14: 656, 1954. 11. Fogh, J.: Reduced tumor-producing capacity of mycoplasma-modified lines of FL human amnion cells in the cheek pouch of cortisonized hamsters. Cancer Res., 29: 1721, 1969. 12. Mitropoulos, K. A. and Balasubramaniam, S.: The role of glucocorticoids in the regulation of the diurnal rhythm of hepatic /3hydroxy-/3-methylglutaryl-coenzyme A reductase and cholesterol 7a-hydroxylase. Biochem. J., 160: 49, 1976.
13. Mitchell, A. D., Pugh, T. D. and Goldfarb, S.: Partial "feedback control" of /3-hydroxy-/3-methylglutaryl coenzyme A reductase activity in primary hepatocellular carcinomas. Cancer Res., 38: 4474, 1978. 14. Siperstein, M. D., Fagan, V. M. and Morris, H.P.: Further studies on the deletion of the cholesterol feedback system in hepatomas. Cancer Res., 26: 7, 1966. 15. Gonzalez, R. and Dempsey, M. E.: Sterol synthesis in cultured human renal cell cancer. J. Urol., 117: 708, 1977. 16. Clayman, R. V., Gonzalez, R., Elliott, A. Y. and Dempsey, M. E.: Differences in cholesterol transport by normal and malignant human kidney cells in culture. Biochem. Biophys. Res. Comm., 92: 1355, 1980. 17. Brown, M. D., Kovanen, P. T. and Goldstein, J. L.: Regulation of plasma cholesterol by lipoprotein receptors. Science, 212: 628, 1981. 18. Brown, M. S., Dana, S. E. and Goldstein, J. L.: Cholesterol ester formation in cultured human fibroblasts. J. Biol. Chem., 250: 4025, 1975. 19. Gonzalez, R., Clayman, R. V. and Dempsey, M. E.: Cholesterol accumulation in renal cancer-a review. Invest. Urol., 19: 1, 1981. 20. Sloan, H. R. and Fredrickson, D. S.: Rare familial diseases with neutral lipid storage. In: The Metabolic Basis of Inherited Disease. Edited by J. B. Stanbury, J. B. Wyngaarden and D. S. Fredrickson. Ed. 3. New York: McGraw Hill, p. 808, 1972. 21. Stein, 0., VanderHoek, J. and Stein, Y.: Cholesterol ester accumulation in cultured aortic smooth muscle cell. Atherosclerosis, 26: 465, 1977.