Expression of P-Glycoprotein in Normal and Malignant Rat Liver Cells

(1)

Expression of P-Glycoprotein in Normal and

Malignant Rat Liver Cells

C . H . LEE,*t G. BRADLEY,*$ AND V. LING*t

*The Ontario Cancer Institute, Princess Margaret Hospital; +Department of Medical Biophysics, University of Toronto; SFaculty of Dentistry, University of Toronto, Toronto, Ontario, Canada, M4X 1K9

Resistance to multiple chemotherapeutic drugs is observed in many advanced human cancers. The molecular basis of such clinical resistance is not understood. One hypothesis is that subpopulations of malignant cells acquire a multidrug-resistant (MDR) phenotype during tumor progression and that such subpopulations have a survival advantage during chemotherapy leading to a nonresponsive disease. Different M D R mechanisms have been identified in cell lines and transplant- able tumors (Morrow and Cowan 1990; Cole et al. 1992; Beck et al. 1993; Gottesman and Pastan 1993; Childs and Ling 1994). One of the best-characterized mechanisms involves the overexpression of the putative drug efflux pump, P-glycoprotein (Pgp) (Endicott and Ling 1989). Pgp has been shown to cause resistance to a variety of unrelated anticancer drugs such as the anthra- cyclines, the vinca-alkaloids, taxol, epipodophyllotox- ins, dactinomycin, and some alkylating agents. Such drugs constitute a significant proportion of our anticancer drugs armamentarium. Thus, it is of interest to determine whether or not the Pgp mechanism of M D R occurs in human cancers.

There are two Pgp genes in humans, MDR1, associated with multidrug resistance, and MDR2/3, not associated with drug resistance but which may be important for phosphatidylcholine translocation in liver (Smit et al. 1993; Ruetz and Gros 1994). Detection of an increased class I Pgp (MDR1) associated with drug resistance can be observed in a wide range of human cancers (Fojo et al. 1987; Goldstein et al. 1989; Arceci 1993). An increased expression of the MDR273 gene in human cancer is rare. There is accumulating evidence that detection of Pgp in tumors at any time during the course of a patient's disease is associated with poor prognosis. In a number of studies, correlation between Pgp expression and nonresponse to chemotherapeutic treatments has been established (Chan et al. 1991; Arceci 1993; Rischin and Ling 1993). In addition, a number of chemosensitizing compounds able to reverse the Pgp-mediated M D R phenotype have been identified (Ford and Hait 1990; Georges et al. 1990). The possibility of using such agents with conventional chemotherapy to improve therapeutic outcome appears promising (Dalton et al. 1989; Sonneveld et al. 1992; List et al. 1993).

Factors important for regulating Pgp expression in human tumors have not been delineated. An increased expression of Pgp in biopsy material obtained from

patients relapsing from chemotherapy has frequently been observed. Such findings are consistent with the hypothesis that subpopulations of Pgp-positive M D R cells are selected by treatment with anticancer drugs. Several studies have also shown that Pgp may be detected in tumor cells of various histological types prior to chemotherapy (Bradley and Ling 1994). The detection of Pgp in tumors at the time of diagnosis often correlates with poor response to subsequent treatment. It has been hypothesized that expression of Pgp in such instances may reflect an association between the M D R phenotype and tumor progression (Bradley and Ling 1994). This hypothesis is supported by studies with children's sarcoma and neuroblastoma, where it was observed that at the time of diagnosis, patients with localized tumors had no detectable Pgp, whereas a significant proportion of nonlocalized tumors showed Pgp-positive cells (Chan et al. 1990, 1991). The incidence of Pgp detected was particularly high for the late-stage tumors, often in metastatic lesions (Chan et al. 1991). The basis for the apparent association of Pgp and tumor progression is not understood.

We have used a well-characterized rat liver carcinogenesis system to investigate systematically the expression of Pgp during tumor progression (Bradley et al. 1992). We examined preneoplastic hepatic foci and nodules, hepatic carcinomas, and lung metastases for expression of Pgp. An increased expression of Pgp during rat liver carcinogenesis was detected initially at the protein level by immunohistochemistry and con- firmed at the m R N A level. We observed no remarkable increase in the level of Pgp in early preneoplastic liver lesions; however, large hyperplastic nodules and hepatocarcinoma showed a heterogeneous increase in Pgp expression. A striking finding was that lung metastases were consistently positive for Pgp. This up-regulation of Pgp appears to involve predominantly one specific Pgp isoform, the class II Pgp. In rats, Pgp is encoded by a small family of three highly homologous genes. In normal liver, the class III Pgp gene not associated with drug resistance is expressed at the highest level. The class I and class II genes which are associated with M D R are expressed at lower levels, with the class II gene being barely detectable. The basis for the increase in class II Pgp expression in rat liver cancer is not understood; however, it may be mechanistically similar to a dramatic increase in the class II Pgp expression observed in cultured rat hepatocytes. In the latter Cold Spring Harbor Symposia on Quantitative Biology, Volume LIX. 9 1994 Cold Spring Harbor Laboratory Press 0-87969-067-4/94 $5.00 607

(2)

608 C.H. LEE, BRADLEY, AND L I N G system, the level of the class II Pgp m R N A increased

more than 20-fold during a 48-hour period, predominantly as a result of an increase in m R N A stability. This increased stability appears to be specific for the class II Pgp, as the other homologous Pgp genes (classes I and III) are not similarly affected. On the other hand, genes unrelated to Pgp, such as actin and tubulin, are up- regulated in this system due also to an increased m R N A stability in this system. Studies using cytoskeleton-disruptive drugs indicate that this increased m R N A stability may be dependent on the integrity of the cytoskeleton. We are currently investigating the possibility that this novel mechanism of m R N A stabilization underlies a regulatory mechanism of broad specificity which may be exploited by tumor cells for malignant progression.

EXPERIMENTAL PROCEDURES

Rat liver carcinogenesis.

Male Fischer 344 rats (Charles River Breeding Laboratories, Wilmington, Massachusetts) weighing 100-120 g were maintained on Purina No. 5001 Rodent Laboratory Chow Diet and daily cycles of alternating 12-hour periods of light and darkness for 1 week before the start of the experiment. Water and food were freely available. Liver carcinogenesis was initiated by a single intraperitoneal (i.p.) injection of dimethylhydrazine at 100 mg/kg, 18 hours after two-thirds partial hepatectomy. After 1 week, the rats were changed from the basal diet to one containing 1% orotic acid (OA) and were maintained on this diet until the time of sacrifice (Fig. 1).

Culture

of primary rat hepatocytes.

Hepatocytes were isolated and cultured as described previously (Lee et al. 1993). In most experiments, unless otherwise stated, hepatocytes were seeded onto type I collagen. No serum or any other additional factors were added to seed the cells. The medium was replaced with fresh medium 4 hours after cell attachment and, thereafter, was replaced daily.

RNA analysis.

Total R N A from cultured cells was isolated by an acid guanidinium isothiocyanate-phenol- chloroform extraction procedure (Chomczynski and Sacchi 1987), and R N A from tissues was extracted with guanidinium thiocyanate followed by centrifugation in cesium chloride solutions (Sambrook et al. 1989). Poly(A) + R N A selection was done using an oligo(dT)- cellulose column (Sambrook et al. 1989). R N A was analyzed either by blot (Northern or slot blot) analysis according to standard procedures (Lee et al. 1993) or by RNase protection, as described by Winter et al. (1985). Blots were hybridized with Pgp gene-specific probes or other random-primed probes as described previously (Lee et al. 1993).

The plasmid constructs used for RNase protection contain a

ThaI-BsaAI

(blunted) 3' rat Pgp2 genomic fragment (positions 120-327) (Deuchars et al. 1992) cloned in

Sinai

(blunted)-digested p G E M 4 Z (Pro- mega) and a

BspMI-DraI

(blunted) 3' rat Pgp3 genomic fragment (positions 113-292) (Deuchars et al. 1992) cloned in

Sinai-digested

pGEM4Z. To synthesize antisense R N A probes, we linearized the Pgp2 and Pgp3 plasmid templates with

EcoRI

(within the vector; transcription with T 7 R N A polymerase). The 3' Pgp2 antisense probe (272 nt) contains 65 nucleotides of vector sequence in addition to a 207-nucleotide

Thai-

BsaAI

fragment of Pgp2. Because a 122-nucleotide sequence within this fragment is identical to that in the

pgpl

m R N A , this probe detects the rat

pgpl

transcript in addition to the rat

pgp2

mRNA. The 3' Pgp3 antisense probe (244 nt) also contains 65 nucleotides of vector sequence in addition to 179 nucleotides from 3' rat genomic Pgp3 and detects a 179-nucleotide fragment of rat

pgp3

mRNA. The pT7 18S template was purchased from Ambion Inc., and when in-vitro-transcribed with T7 R N A polymerase, it produced a 109- nucleotide antisense probe, of which 80 nucleotides is complementary to 18S ribosomal RNA. Labeled R N A was hybridized with 30/xg of total RNA, and protected fragments were visualized by electrophoresis through a

w+oL

I

Altered

Small

Large

hepatic

hyperplastic

hyperplastie

loci

nodules

i

60wks.

I

Hepatocellular

carcinoma

Lung

metastases

Figure 1. Protocol for rat liver carcinogenesis showing sequential development of preneoplastic liver lesions and liver cancer over a period of - 6 0 weeks. (PH) Two-thirds partial hepatectomy; (DMH) dimethylhydrazine dihydrochloride; (OA) orotic acid.

(3)

denaturing 6% polyacrylamide gel, followed by autoradiography.

Immunohistochemical

detection

of

Pgp.

Immunohistochemical staining for Pgp was performed on frozen sections fixed for 10 minutes in cold acetone. After rinsing with phosphate-buffered saline (PBS), endogenous peroxidase activity and endogenous biotin- like activity were blocked by appropriate pretreat- ments, and immunohistochemical staining was carried out as described previously (Bradley et al. 1990). Anti- body C219 was used at 1.5/xg/ml in 1% bovine serum albumin (BSA) in an overnight incubation (16 hr) at 4~

RESULTS Rat Liver Carcinogenesis

Rat liver carcinogenesis models are attractive for investigating cellular and molecular events during tumor development.Various models of rat liver carcinogenesis have been developed in which sequential steps of the carcinogenic process can be observed, and some of the histological and biochemical characteristics of these sequential events have been elucidated (Farber and Cameron 1980). Analysis of Pgp expression during the stepwise process of liver cancer development in such models may lead to a better understanding of the relationship between increased Pgp expression and the carcinogenic process. We have used the OA model of rat liver carcinogenesis initiated by partial hepatectomy coupled with an injection of dimethylhydrazine (DMH) followed by 50-60 weeks of promotion with a 1% OA diet (Fig. 1). Close to 100% of the animals developed hepatocarcinoma, and the sequential development of hepatic loci, hyperplastic nodules, and liver carcinoma can be observed over this period. Between 30% and 60% of the animals develop metastases to the lungs (Laurier et al. 1984; Bradley et al. 1992). Using histological staining to follow the progressive development of hepatic lesions in this system, it may be seen that after 5 weeks, hepatic foci were detectable by increased levels of 3,-glutamyl transpeptidase (GGT) and glutathione S-transferase 7.7 (GST-P). These en- zymes are thought to be involved in hepatic detoxifica- tion pathways. There was no detectable change in Pgp expression as stained with monoclonal antibody C219 in the hepatic foci. The hepatocytes of the foci dis- played polarized staining for P-glycoprotein on the bile canalicular face of the plasma membrane similar to that observed in normal liver. In the small hyperplastic nodules ( < 1 cm in diameter), a heterogeneous pattern of Pgp staining was observed. The majority of the hepatocytes showed little or no Pgp staining; this was interspersed with groups of hepatocytes which stained strongly for Pgp. Large hyperplastic nodules (1 cm or more in diameter) showed stronger staining for Pgp, although there was again notable heterogeneity in Pgp expression within each nodule and among nodules. In

these nodules, an association was observed between the presence of prominent vascular sinusoids separating trabeculae of hepatocytes and high levels of Pgp staining. After 50 or more weeks of promotion, the liver was typically occupied by a large, irregular tumor. Within the liver tumors, areas with moderate to strong Pgp staining were observed, and intense staining for Pgp was often found in areas of trabecular cancer with wide intervening sinusoids (Fig. 2). Pgp expression was consistently observed in lung metastases. Of more than 400 metastases examined, every metastasis contained cells that were positive for Pgp, regardless of its size. The surrounding lung tissue did not stain for Pgp (Fig. 2). The above data suggest that an increased expression of Pgp is associated with relatively late stages in rat liver carcinogenesis. Similar findings were obtained in a study of Pgp m R N A expression by in situ hybridization of preneoplastic and neoplastic liver lesions induced by the Solt-Farber model of hepatocarcinogenesis (Nakat- sukasa et al. 1992). The pattern of Pgp overexpression thus appears to be distinct from that of GST-P and GGT, which are commonly used markers of rat liver carcinogenesis and are clearly overexpressed in the earliest foci of altered hepatocytes. Increased expression of Pgp may be one of the molecular events that contribute to the growth of hyperplastic nodules and, subsequently, liver cancer. The data from the immunohistochemical study also suggest a possible role for Pgp in the vascularization of preneoplastic liver nodules and liver cancer, and in the process of hematogenous metastasis to the lungs. Direct investigations into these possibilities may further our understanding of the role of Pgp in carcinogenesis.

Expression of P-Glycoprotein Isoforms in Rat Liver Cancers

P-Glycoprotein in rat is encoded by three Pgp genes (Deuchars et al. 1992). In mammalian cells, the class I and II Pgp genes confer multidrug resistance, whereas the class III gene does not. These three classes of genes are expressed differentially in normal tissues, usually with one class predominating (Croop et al. 1989; Brad- ley et al. 1990). These findings suggest that Pgp isoforms have distinct physiological roles associated with specialized cell functions. In the rat liver, the class III isoform predominates, although the class I Pgp is also detectable. The class II Pgp is not readily detectable by standard techniques.

To determine what Pgp isoform might be expressed during rat liver carcinogenesis, we employed the 3' untranslated region of rat Pgp genes to differentiate between the expression of the three classes of Pgp genes. Currently, antibodies specific for each of the rat Pgp isoforms are not available. As shown in Figure 3, the class III Pgp mRNA is expressed at the highest level in normal rat liver. We estimate that the class I Pgp gene is expressed at about 10% of the class III Pgp gene, whereas class II Pgp expression is barely detectable in this assay. When a number of different hepato-

(4)

610 C.H. LEE, BRADLEY, AND LING

Figure 2. lmmunoperoxidase staining for P-glycoprotein using C219 as primary antibody, diaminobenzidine as the final substrate, and hematoxylin as nuclear counterstain. (Top left) Normal rat liver, showing Pgp on the bile canalicular face of the hepatocytes. Magnification, 125 X. (Top right) Primary hepatocarcinoma, showing overexpression of Pgp which remains predominantly in a polarized membrane distribution. Magnification, 125 • (Bottom left) Multiple metastases of hepatocarcinoma in the lungs, showing Pgp in the metastases but not in the surrounding lung. Magnification, 20 • (Bottom right) High-power view of one of the metastases in bottom left panel, showing strong staining for Pgp in many of the metastatic cells. Magnification, 125 •

L L T T S

W

•

~ 3

Figure 3. Northern blot analysis of expression of the three Pgp genes (pgp 1, 2, and 3) in normal and malignant rat liver. Lanes marked L indicate two separate samples of normal male rat liver. Lanes marked T are two samples taken from different areas of a large liver carcinoma. Lane S is a sample taken from nontumorous liver in the same rat as T. Each lane has been loaded with 10/xg of poly A + RNA and the blots were probed with gene-specific oligonucleotide probes derived from the 3'-untranslated region of each pgp gene.

carcinomas were examined, there appeared to be a dramatic increase in the class II Pgp gene expression, as shown in Figure 3. There is no comparable increase in expression of class I and class III Pgp in the hepato- carcinomas. A comparison of the findings by immunohistochemical staining and those by Northern blot analysis of a large number of preneoplastic liver lesions and liver cancer suggest that overexpression of the class II Pgp isoform must account for virtually all the increase in immunohistochemical staining for Pgp during rat liver carcinogenesis. The specific and marked overexpression of one Pgp isoform (class II Pgp) during rat hepatocarcinogenesis has been reported for different models of liver carcinogenesis (Teeter et al. 1993; Fardel et al. 1994), supporting the hypothesis that this aberrant pattern of expression of the Pgp isoforms is characteristic of the hepatocarcinogenic process itself, irrespective of the agents used for carcinogenesis. At present, it is not known by what mechanism the class II P-glycoprotein is turned on during rat hepatocarcinogenesis. It is clear, however, that the up-regulation of Pgp in this process appears to be specific for the class II gene, as the class I and class III genes are not similarly affected.

In various systems, P-glycoprotein expression appears to be highly responsive to a variety of factors. Treatment of cultured cells with a wide range of chemi-

(5)

cals, as well as heat shock or X-irradiation, can lead to an increase in Pgp expression (Kohno et al. 1989; Arceci et al. 1990; Chin et al. 1990; Hill et al. 1990). In some systems, treatment of cell lines with so-called differentiating reagents (such as sodium butyrate or retinoic acid) results in increased expression of Pgp (Bates et al. 1989; Mickley et al. 1989). The transcription of the Pgp gene may be increased through the activation of specific oncogenes. The

c-Ha-ras-1

oncogene and a mutant

p53

gene have been shown to stimulate activity of the human MDR1 promoter in NIH-3T3 cells (Chin et al. 1992). In another study, a mutant murine

p53

gene was found to stimulate the activity of the hamster Pgpl promoter which had been transfected into Chinese hamster ovary cells, whereas the wild-type

p53

gene suppressed its activity (Zastawny et al. 1993). Although mutations in the Ha-

ras

oncogene and the

p53

gene have been identified in hepatocellular carcinomas, we do not know whether or not such mutations have occurred in our system or if they affect Pgp expression. Such studies raise several possibilities for the mechanism of Pgp overexpression during carcinogenesis. One possibility is that malignant cells activate in an aberrant manner physiological pathways which cause elevated Pgp expression that are not normally seen in a particular tissue; for example, a high level of class II Pgp in liver. Alternatively, the tumor cells may be exposed to external factors that trigger an increased expression of Pgp. Studies to investigate these possible mechanisms of Pgp expression in tumor cells will contribute significantly to our understanding of molecular alterations associated with tumor progression.

Expression of Pgp Genes in Primary Rat Hepatocytes

Primary rat hepatocytes exhibit many properties of normal liver, and they have been used as a model system for investigating liver function and gene expression (Berry et al. 1991). We and other workers have investigated Pgp expression in primary hepatocyte cultures under different conditions (Fardel et al. 1992; Lee et al. 1993; Schuetz and Schuetz 1993). It was observed that under a simple culture even in the absence of serum, there was a dramatic increase in the level of the class II Pgp

(pgp2)

expressed upon culturing of freshly isolated hepatocytes (Fig. 4). After 48 hours in culture, there was a greater than 20-fold increase in the level of

pgp2

m R N A detected. The levels of the class I Pgp m R N A

(pgpl)

also increased in hepatocyte cultures, but the change was clearly less significant than that of

pgp2

(Fig. 4). In contrast, the most abundant Pgp in the liver, the class III Pgp

(pgp3),

exhibited a decline. These results indicated a differential regulation of the different Pgp genes and that the primary hepatocytes provide an opportunity to further dissect these mechanisms. Significantly, a dramatic increase in

pgp2

expression is also observed during rat liver carcinogenesis

I 217- 2 0 t - 190- 1 8 0 - 1 6 0 - 147-

]PgP3

1 2 3 - 1 1 0 - 9 0 - ~S

Figure 4. RNase protection analysis of total RNA from rat liver, 48-hr cultured rat hepatocytes, and rat hepatoma RC3 cells. Autoradiography showing the protected fragments of the

pgp2

(207 nt),

pgpl

(122 nt), and

pgp3

(179 nt) transcripts. The transfer RNA control hybridization in each panel is shown. As size marker, an end-labeled

HpaII

digest of pBBR322 DNA was used.

(Fig. 3), suggesting that a similar mechanism may be operating in these two systems.

Changes in the expression of other genes are also observed in hepatocyte cultures. Although less dramatic than

pgp2,

the expression of cytoskeletal genes (a-tubulin and fl-actin) was also found to increase with time in culture (Fig. 5). This increase in m R N A level is specific only for a subset of mRNAs, as m R N A for other genes such as albumin (data not shown) and connexin32 (Fig. 5) decreased as a function of time in culture. Further correlation between

pgp2

and cytoskeletal (actin and tubulin) gene expression was observed in a variety of culture conditions, suggesting that there may be a common mechanism regulating these apparently unrelated genes (Lee et al. 1993). Changes in the expression of these genes also appear to be closely associated with the cell shape of hepatocytes. For instance, hepatocytes cultured on matrigel were round, compact, and expressed significantly lower levels of

pgp2

than hepatocytes cultured on collagen-coated dishes (Fig. 6). Since cell shape is determined to a large extent by the organization of cytoskeletal network, we examined the importance of an intact cytoskeleton in the increased

pgp2

expression. We disrupted the hepatocyte microfilaments and micro- tubules with cytochalasin D and colchicine, respectively. As shown in Figure 7, the gradual increase in

pgp2

expression was greatly reduced in the presence of either drug, suggesting that the increase in

pgp2

expression in

(6)

612 C.H. LEE, BRADLEY, AND L I N G

D

- 2 8 8

-1811

Figure 5. Northern analysis of mRNAs from rat liver, isolated rat hepatocytes, and cultured hepatocytes. Each lane contained 20 #g of total RNA from liver, isolated hepatocytes (I.H.), and hepatocytes cultured for various lengths of time as indicated. The same membrane was sequentially stripped and reprobed for a-tubulin (panel B), fl-actin (panel C), and connexin 32 (panel D). Ethidium-bromide-stained gel at the bottom of the figure confirms that approximately equal amounts of RNA were loaded per lane. (Adapted from Lee et al. 1993.)

hepatocyte cultures is dependent on the integrity of the cytoskeleton. It is interesting to note that the parallel rise in m R N A levels for both actin and tubulin was also significantly affected.

Stability of

pgp2

mRNA in Primary Rat

Hepatocytes

The underlying mechanism for the large increase in

pgp2

expression in primary rat hepatocytes was investigated. Nuclear run-on studies were conducted to direct- ly measure the transcription rate of

pgp2

as a function of time in culture. We could not detect any significant change in

pgp2

transcription that would parallel the change in m R N A levels (C.H. Lee et al., in prep.). We then set out to establish whether

pgp2

m R N A stability is altered. Transcription was arrested in hepatocyte cultures at several time points by actinomycin D at

P-gp 2

6 - a c t l n

Of.- tubulln

~ ? ~ii~i~i!~!! i~

/i-,

Figure 6. Morphology

(left

panel) and mRNA expression

(right

panel) of hepatocytes cultured on collagen-coated dishes and matrigel. Northern analysis of mRNAs from hepatocytes cultured for 48 hr on collagen-coated dishes or matrigel

(right

panel). Each lane contains 20 /zg total RNA, and ethidium bromide stain of gel is shown at the bottom of the figure. The

left

panel shows morphology of hepatocytes cultured for 24 hr on collagen-coated dishes and matrigel. Phase contrast, magnification 175 • (Adapted from Lee et al. 1993.)

concentrations effective in inhibiting more than 98% of new R N A synthesis,

pgp2

m R N A was then followed over a 16-hour period by slot blot analysis to determine its half-life. The half-life of the

pgp2

transcript for 8-, 12-, 36-, and 48-hour cultures is approximately 4.5, 6, 13, and 16 hours, respectively (C.H. Lee et al., in prep.). A linear relationship is obtained when the log of relative

pgp2

m R N A content is plotted against the half-life of

pgp2

m R N A (Fig. 8). This indicated that the dramatic elevation of

pgp2

m R N A in cultured hepatocytes is paralleled quantitatively by a dramatic increase in m R N A half-life. A n increase in m R N A stability thus appears to be responsible for the elevation of

pgp2

m R N A in primary rat hepatocyte cultures.

Since the data in Figures 6 and 7 suggest that cytoskeletal components are involved in regulating

pgp2

expression in the hepatocyte culture, we determined if the stability of

pgp2

m R N A was different in the presence or absence (control) of cytochalasin D. In a 36- hour hepatocyte culture, the half-life of

pgp2

transcript in control cultures was approximately 14 hours, but it was 5 hours in the presence of cytochalasin D (Fig. 9). These results therefore indicated that the reduced

pgp2

transcript levels seen in the presence of cytochalasin D (Fig. 7) are most likely due to a decrease in stability of the m R N A .

(7)

O h 2 4 h

Figure 7. Effects of colchicine and cytochalasin D on class If Pgp expression. After 4 hr for cell attachment, cultures were either harvested for total RNA (None, 0 h) or changed to fresh medium containing no addition (None, 24 hr), 0.6 /zg/ml colchicine, or 0.8/zg/ml cytochalasin D. After a further 24-hr incubation, cultures were harvested for total RNA. Each well had 20/zg total RNA, and ethidium bromide stain of gel is shown at the bottom of the figure to indicate equal loading of RNA. (Adapted from Lee et al. 1993.)

D I S C U S S I O N

H u m a n cancers are composed of heterogeneous populations of tumor cells, of which only a portion are considered to be malignant stem cells capable of self- renewal and progression. A major challenge in cancer biology is to identify molecular alterations associated with malignant progression and to delineate the role such molecules play. Accumulating evidence suggests that detection of P-glycoprotein in tumor biopsies co- incides with a therapy-resistant disease in a variety of human cancers. There is little controversy that overexpression of Pgp in malignant cells would confer resistance to many anticancer drugs and thus provide such cells with a growth advantage during treatment with chemotherapy. What additional roles Pgp may play during tumor progression are not understood. Pgp is likely to transport a broad range of substrates in addition to anticancer drugs (Gottesman and Pastan 1993; Childs and Ling 1994). These substrates may include steroid hormones, peptides, A T E and ions. It is possible that in some cancers, the overexpression of Pgp provides growth advantages to the tumor cells through secretion of autocrine growth factors, removal of toxic metabolites, or secretion of angiogenic factors to promote vascularization. In support of such a hypothesis is the observation that an increased level of Pgp is frequently detected in advanced human cancers at diagnosis prior to chemotherapy. This observation in human cancers is paralleled by findings in the rat liver carcinogenesis system, where overexpression of Pgp occurred during late stages of carcinogenesis and where

i i i 3 0 2 0 m D m < 10 Z E tN O. 01 O. I II I I I I | I j ,

I

2

4 8 h e 3 6 h 0 j

8h e 1 ~ , p ~

u 1 i I I i a i i i I i , i i J i 4 6 8 10 12 14 16 H a l f - l i f e ( h )

Figure 8. Linear relationship between the log of relative class II Pgp mRNA content and half-life. Relative pgp2 mRNA content is obtained from multiple experiments, including those in Fig. 5. The half-lives of pgp2 mRNA at different culture times were obtained by arresting transcription with 5/~g/ml actinomycin D, and following the decay of the transcripts, by slot blot analysis. Extrapolation of data is shown by broken line.

(8)

614 C.H. LEE, BRADLEY, AND LING

A)

T , . .

Act D addition

(hours)

O-

1 - . ~ D , 9 i 6 - m 1 2 - ~ / ~ _---.. 1 9 - m

\

o.o,

I !

'%

, ;

,.

20 Time (hours)

Figure 9. Rapid decay of pgp2 mRNA in the presence of cytochalasin D. After 36 hr in culture, hepatocytes were treated with 5 #g/ml actinomycin D in the presence or absence (control) of 0.8/xg/ml cytochalasin D. Total RNA (10/zg) was subjected to slot blot analysis as shown (A). (B) Results of densitometry of the data in A. Error bars represent the range of two independent experiments using different hepatocyte preparations.

metastases to the lungs consistently expressed a high level of Pgp. Future studies involving inactivation of Pgp function or down-regulation of Pgp expression may provide further insights into the different roles Pgp may play in chemotherapy and during tumor progression.

How P-glycoprotein is regulated in human cancers is not understood; however, insights into this question may reveal factors important for mediating expression of Pgp and other malignancy-associated genes. Such factors, along with Pgp, may be useful targets for modulation to improve therapeutic efficacy. We have used a well-characterized rat liver carcinogenesis system to systematically examine the association between Pgp expression and carcinogenesis. The fact that the class II Pgp is highly overexpressed in rat liver tumors argues against a model in which tumor cells are simply expressing a differentiated phenotype of their tissue of origin, since the class II Pgp is usually not detectable in liver. Rather, the level of Pgp observed in rat liver tumor cells during carcinogenesis represents an aberrant up-regulation of an MDR-associated Pgp gene.The fact that in cultured rat hepatocytes the class II Pgp gene was also dramatically up-regulated suggests that this latter system may be a model for understanding the regulation of Pgp in rat liver tumor cells. The findings that the increased level of the class II Pgp gene of hepatocytes in culture is due to a dramatic increase in m R N A stability, that this mechanism is dependent on the integrity of the cytoskeleton, and that other non- Pgp-related genes such as actin and tubulin may be regulated in a similar manner raise the possibility that this is a novel mechanism which coordinately affects many apparently unrelated genes. We speculate that such a mechanism which is capable of regulating the expression of multiple genes, including Pgp, through m R N A stabilization may play an important role in the development of malignant disease.

A C K N O W L E D G M E N T S

We thank our colleagues at the Ontario Cancer Institute for helpful discussions. This work was supported by grants from the National Cancer Institute of Canada.

R E F E R E N C E S

Arceci, R. 1993. Clinical significance of P-glycoprotein in multidrug resistance malignancies. Blood 81: 2215. Arceci, R.J., F. Baas, R. Raponi, S.B. Horwitz, D. Housman,

and J.M. Croop. 1990. Multidrug resistance gene expression is controlled by steroid hormones in the secretory epithelium of the uterus. MoL Reprod. Dev. 25: 101. Bates, S.E., L.A. Mickley, Y. Chen, N. Richert, J. Rudick, J.L.

Biedler, and A.T. Fojo. 1989. Expression of a drug resistance gene in human neuroblastoma cell lines: Modulation by retinoic acid-induced differentiation. Mol. Cell. Biol. 9: 4337.

Beck,W.T., M.K. Danks, J.S.Wolverton, R. Kim, and M. Chen. 1993. Drug resistance associated with altered DNA topoisomerase II. Adv. Enzyme Regul. 33: 113.

Berry, M.N., A.M. Edwards, and G.J. Barritt. 1991. Monolayer culture of hepatocytes. In Isolated hepatocytes preparation, properties, and applications (ed. R.H. Burdon and P.H. Van

Knippenberg), p. 265. Elsevier, The Netherlands. Bradley, G. and V. Ling. 1994. P-glycoprotein, multidrug

resistance and tumor progression. Cancer Metastasis Rev. 13: 223.

Bradley, G., E. Georges, and V. Ling. 1990. Sex-dependent and independent expression of the P-glycoprotein isoforms in Chinese hamsters. J. Cell Physiol. 145: 398.

Bradley, G., R. Sharma, S. Rajalakshmi, and V. Ling. 1992. P-glycoprotein expression during tumor progression in the rat liver. Cancer Res. 52: 51.

Chan, H.S.L., P.S. Thorner, G. Haddad, and V. Ling. 1990. Immunohistochemical detection of P-glycoprotein: Prog- nostic correlation in soft tissue sarcoma of childhood. J. Clin. Oncol. 8:689

Chan, H.S.L., G. Haddad, P.S. Thorner, G. DeBoer, Y.P. Lin, N. Ondrusek, H. Yeger, and V. Ling. 1991. P-glycoprotein expression as a predictor of the outcome of therapy for neuroblastoma. N. Engl. J. Med. 325: 1608.

(9)

Childs, S. and V. Ling. 1994. The M D R superfamily of genes and its biological implications. In Important advances in oncology (ed. V.T. DeVita et al.), p. 21. J.B. Lippincott, Philadelphia, Pennsylvania.

Chin, K.V., K. Ueda, I. Pastan, and M.M. Gottesman. 1992. Modulation of activity of the promoter of the human MDR1 gene by Ras and p53. Science 255: 459.

Chin, K.V., S. Tanaka, G. Darlington, I. Pastan, and M.M. Gottesman. 1990. Heat shock and arsenite increase expression of the multidrug resistance (MDR1) gene in human renal carcinoma cells. J. Biol. Chem. 265: 221.

Chomczynski, E and N. Sacchi. 1987. Single-step method of R N A isolation by acid guanidinium thiocyanate-phenol- chloroform extraction. Anal. Biochem. 162" 156.

Cole, S.RC., G. Bhardwaj, J.H. Gerlach, J.E. Mackie, C.E. Grant, K.C. Almquist, A.J. Stewart, E.U. Kurz, A.M.V. Duncan, and R.G. Deeley. 1992. Identification of a novel ATP-binding cassette transporter gene overexpressed in a multidrug resistant human lung cancer cell line. Science 258:1650.

Croop, J.M., M. Raymond, D. Haber, A. Devault, R.J. Arceci, E Gros, and D.E. Housman. 1989. The three mouse multidrug resistance (mdr) genes are expressed in a tissue specific manner in normal mouse tissues. Mol. Cell. Biol. 9: 1346.

Dalton, W.S, T.M. Grogan, RS. Meltzer, R.J. Scheper, B.G.M. Durie, C.W. Taylor, T.E Miller, and S.E. Salmon. 1989. Drug-resistance in multiple myeloma and non-Hodgkin's lymphoma: Detection of P-glycoprotein and potential cir- cumvention by addition of verapamil to chemotherapy. J. Clin. Oncol. 7: 415.

Deuchars, K.L., D. Duthie, and V. Ling. 1992. Identification of distinct P-glycoprotein gene sequences in rat. Biochim. Biophy. Acta 1130: 157.

Endicott, J.A. and V. Ling. 1989. The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu. Rev. Bio- chem. 58: 137.

Father, E. and R. Cameron. 1980. The sequential analysis of cancer development. Adv. Cancer Res. 31: 125.

Fardel, O., E Loyer,V. Lecureur, D. Glaise, and A. Guillouzo. 1994. Constitutive expression of functional P-glycoprotein in rat hepatoma cells. Eur. J. Biochem. 219: 521. Fardel, O., R Loyer, F. Morel, D. Ratanasavanh, and A.

Guillouzo. 1992. Modulation of multidrug resistance gene expression in rat hepatocytes maintained under various culture conditions. Biochem. Pharmacol. 44: 2259. Fojo, A.T., K. Ueda, D.J. Salmon, D.G. Poplack, M.M. Gottes-

man, and I. Pastan. 1987. Expression of a multidrug-resistance gene in human tumors and tissues. Proc. Natl. Acad. Sci. 84: 265.

Ford, J.M. and W.N. Hait. 1990. Pharmacology of drugs that alter multidrug resistance in cancer. Pharmacol. Rev. 42:155.

Georges, E., F.J. Sharom, and V. Ling. 1990. Multidrug resistance and chemosensitization: Therapeutic implications for cancer chemotherapy. Adv. Pharmacol. 21: 185.

Goldstein, L.J., H. Galski, A. Fojo, M. Willingham, A.-L. Lai, A. Gazdar, R. Pirker, A. Green, W. Crist, G.M. Brodeur, M. Lieber, J. Cossman, M.M. Gottesman, and I. Pastan. 1989. Expression of a multidrug resistance gene in human tumors. J. Natl. Cancer Inst. 81: 116.

Gottesman, M.M. and I. Pastan. 1993. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem. 62: 385.

Hill, B.T., K. Deuchars, L.K. Hosking, V. Ling, and R.D. Whelan. 1990. Overexpression of P-glycoprotein in mam-

malian tumor cell lines after fractionated X irradiation in vitro. J. Natl. Cancer Inst. 82: 607.

Kohno, K., S. Sato, H. Takano, K. Matsuo, and M. Kuwano. 1989. The direct activation of human multidrug resistance gene (MDR1) by anticancer agents. Biochem. Biophys. Res. Commun. 165: 1415.

Laurier, C., M. Tatematsu, EM. Rao, S. Rajalaksmi, and D.S.R. Sarma. 1984. Promotion by orotic acid of liver carcinogenesis in rats initiated by 1,2-dimethylhydrazine. Cancer Res. 44: 2186.

Lee, C.H., G. Bradley, J.-T. Zhang, and V. Ling. 1993. Dif- ferential expression of P-glycoprotein genes in primary rat hepatocyte culture. J. Cell. Physiol. 157: 392.

List, A.F., C. Spier, J. Greer, S. Wolf, J. Hutter, R. Dorr, S. Salmon, B. Futcher, M. Baier, and W. Dalton. 1993. Phase I / I I trial of cyclosporine as a chemotherapy-resistant modi- fier in acute leukemia. J. Clin. Oncol. 11: 1652.

Mickley, L.A., S.E. Bates, N.D. Richert, S. Currier, S. Tanaka, F. Foss, N. Rosen, and A.T. Fojo. 1989. Modulation of the expression of a multidrug resistance gene (mdr-1/P-glycoprotein) by differentiating agents. J. Biol. Chem. 264: 18031.

Morrow, C.S. and K.H. Cowan. 1990. Glutathione-S-transfer- ases and drug resistance. Cancer Cells 2: 15.

Nakatsukasa, H., R.E Evarts, R.K. Burt, E Nagy, and S.S. Thorgeirsson. 1992. Cellular pattern of multidrug-resistance gene expression during chemical hepatocarcinogenesis in the rat. Mol. Carcinog. 6: 190.

Rischin, D. and V. Ling. 1993. Multidrug resistance in leukemia. In Leukemia: Advances, research and treatment (ed. E J . Freireich and H. Kantarjian), 269. Kluwer Aca- demic, The Netherlands.

Ruetz, S. and E Gros. 1994. Phosphatidylcholine translocase: A physiologic role for the mdr2 gene. Cell 77: 1071. Sambrook. J., E.F. Fritsch, and T. Maniatis. 1989. Molecular

cloning: A laboratory manual, 2nd edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Schuetz, J.D. and E.G. Schuetz. 1993. Extracellular matrix

regulation of multidrug resistance in primary monolayer cultures of adult rat hepatocytes. Cell Growth Differ. 4: 31. Smit, JJ.M., A.H. Schinkel, R.EJ. Oude Elferink, A.K. Groen, E. Wagenaar, L. Van Deemter, C.A.A.M. Mol, R. Ot- tenhofer, N.M.T. Van der Lugt, M.A. Van Roon, M.A. Van der Valk, G J . A . Offerhaus, A.J.M. Berns, and E Borst. 1993. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 75: 451.

Sonneveld, E, B.G.M. Durie, H.M. Lokhorst, J. Marie, G. Solbu, S. Suciu, R. Zittoun, B. Lowenberg, and K. Nooter. 1992. Modulation of multidrug-resistant multiple myeloma by cyclosporin. Lancet 340: 255.

Teeter, L.D., M. Estes, J.Y. Chart, H. Atassi, S. Sell, F.F. Becker, and M.T. Kuo. 1993. Activation of distinct multidrug-resistance (P-glycoprotein) genes during rat liver regeneration and hepatocarcinogenesis. Mol. Carcinog. 8: 67.

Winter, E., F. Yamamoto, C. Almoguera, and M. Perucho. 1985. A method to detect and characterize point mutations in transcribed genes: Amplified and overexpression of the mutant c-Ki-ras allele in human tumor cells. Proc. Natl. Acad. Sci. 82: 7575.

Zastawny, R.L., R. Salvino, J. Chen, S. Benchimol, and V. Ling. 1993. The core promoter region of the P-glycoprotein gene is sufficient to confer differential responsiveness to wild-type and mutant p53. Oncogene 8: 1529.

(10)

10.1101/SQB.1994.059.01.069

Access the most recent version at doi:

1994 59: 607-615

Cold Spring Harb Symp Quant Biol

C.H. Lee, G. Bradley and V. Ling

Liver Cells

Expression of P-Glycoprotein in Normal and Malignant Rat

References

http://symposium.cshlp.org/content/59/607.full.html#ref-list-1

This article cites 40 articles, 16 of which can be accessed free at:

License

Commons

Creative

Service

Email Alerting

click here.

the box at the top right corner of the article or

Receive free email alerts when new articles cite this article - sign up in

http://symposium.cshlp.org/subscriptions

go to: Cold Spring Harbor Symposia on Quantitative Biology To subscribe to