The use of a novel taxane-based P-glycoprotein inhibitor to identify mutations that alter the interaction of the protein with paclitaxel.

Murine thymoma cell lines expressing mutated forms of the mdr1b P-glycoprotein were isolated using a novel taxane-based P-glycoprotein inhibitor tRA-96023 (SB-RA-31012). The selection strategy required resistance to a combination of tRA-96023 and colchicine. Five mutations were identified (N350I, I862F, L865F, L868W, and A933T) that reduce the capacity of tRA-96023 to inhibit P-glycoprotein-dependent drug resistance. These mutations also result in a loss of paclitaxel resistance ranging from 47 to 100%. Four mutations are located in the second half of the protein, within or near the proposed transmembrane segment (TMS) 10--11 regions. The fifth mutation (N350I) is within the first half of the protein, proximal (cytoplasmic) to TMS 6. The variant cell line expressing the L868W mutation was subjected to a second round of selection involving tRA-96023 and the toxic drug puromycin. This resulted in the isolation of a cell line expressing a P-glycoprotein with a double mutation. The additional mutation (N988D) is located within TMS 12 and conveys further decreases in resistance to paclitaxel and the capacity of tRA-96023 to inhibit drug resistance. Taken together, the results indicate a significant contribution by the TMS 10--12 portion of the protein to the recognition and transport of taxanes and give evidence that the cytoplasmic region proximal to TMS 6 also plays a role in taxane interactions with P-glycoproteins. Interestingly, mutations within TMS 6 and 12 were found to cause a partial loss of PSC-833 inhibitor activity, suggesting that these regions participate in the interactions with cyclosporin and its derivatives.

Profound differences in the transport of steroids by two mouse P-glycoproteins.

There are two mouse P-glycoproteins that convey multidrug resistance, mdr1 (mdr1b) and mdr3 (mdr1a), by serving as drug efflux transporters. These proteins each exhibit tissue-specific expression. There is relatively high expression of the mdr1 gene in the adrenals, the site of glucocorticoid and mineralocorticoid hormone synthesis. We previously demonstrated that mdr1 gene expression in murine thymoma cells correlated well with a decrease in their ability to accumulate the glucocorticoid dexamethasone and their increased resistance to glucocorticoid-induced apoptosis. Additional evidence is presented that supports the proposition that the mdr1 P-glycoprotein can transport glucocorticoids. Specifically, introduction and expression of the mouse mdr1 gene in the human HEK 293T cell line conveys a multidrug resistance phenotype that includes a reduced capacity to accumulate dexamethasone. Moreover, isolation of additional mdr1-expressing mouse lymphoid cells, without using steroids in the selection, confirms the linkage between multidrug resistance conveyed by the mdr1 P-glycoprotein and resistance to dexamethasone. In contrast, two newly isolated lymphoid lines, selectively expressing the mdr3 gene, were not found to have increased dexamethasone resistance or the capacity to accumulate significantly lower levels of hormone. The results support the concept that the mdr1 and mdr3 P-glycoproteins may serve alternative roles in the transport of endogenous substances such as steroids.

Identification of P-glycoprotein mutations causing a loss of steroid recognition and transport.

P-glycoproteins transport a wide variety of hydrophobic compounds out of cells. While the diversity of transported molecules suggests a mechanism involving broad specificity, there is evidence of significant discrimination within given classes of molecules. One example of this behavior is transport of corticosteroids by the murine mdr1 P-glycoprotein. The presence of hydroxyl groups, associated with specific steroid carbon atoms, regulates the ability of corticosteroids to be transported. This specificity is demonstrated here by experiments measuring the ability of steroids to inhibit drug transport. The results indicate that a keto oxygen associated with the 3- and 20-carbon atoms, as well as a 17-carbon hydroxyl group, each acts to enhance steroidal P-glycoprotein inhibitory activity. Moreover, inhibitory steroids can be used for directed selection of variant cells, expressing mutated P-glycoproteins with a severely impaired ability to transport dexamethasone. The five mutations, reported here, are located within transmembrane domains 4-6, proximal to the cytoplasmic interface. The altered P-glycoproteins exhibit reduced capacity to be inhibited by specific steroids, suggesting decreased capacity to bind these molecules avidly. Studies comparing the relative inhibitory activity of a series of steroids indicate that these mutations alter recognition of the 17alpha-hydroxyl group and the 20-keto oxygen atom.

Chemosensitizing steroids: glucocorticoid receptor agonists capable of inhibiting P-glycoprotein function.

P-glycoprotein expression in lymphoid malignancies has the potential to compromise the efficacy of many therapeutic regimens using anthracyclines, glucocorticoids, and Vinca alkaloids. All three classes of drugs are transported by P-glycoproteins. We have explored the possibility that modified steroids could serve a dual purpose, as glucocorticoid receptor agonists and P-glycoprotein inhibitors. Substitution of such steroids for those currently in use would help to overcome the selective advantage held by cells expressing P-glycoproteins. 17-Deoxydexamethasone and dichlorisone were modified by the addition of a dimethylamino benzoate group at the 21-carbon atom of the steroids. The two resulting steroids, SA47 and SA450, were potent glucocorticoid receptor agonists also capable of inhibiting the human P-glycoprotein with an efficiency equal to that of verapamil. Thus, both compounds are examples of steroids that could potentially serve as beneficial substitutions for dexamethasone or prednisolone in the chemotherapy of lymphomas and leukemias.

Expression of the mdr1 P-glycoprotein gene: a mechanism of escape from glucocorticoid-induced apoptosis.

Glucocorticoid hormones cause apoptosis in the murine T-lymphoma cell line WEHI-7. Glucocorticoid receptors in these cells are cytoplasmic proteins that translocate to the nucleus upon binding hormone. Thus, regulation of cytoplasmic glucocorticoid concentrations controls the level of activated receptors and sensitivity to steroid-induced apoptosis. We found that expression of the mdr1 P-glycoprotein gene produces a reduced accumulation of dexamethasone in WEHI-7 cells. Concomitantly, there is a suppression of dexamethasone-induced changes in transcription and a decrease in steroid sensitivity. P-glycoproteins are known to cause an outward, ATP-dependent transport of a variety of unrelated hydrophobic drugs across the plasma membrane. Our results indicate that glucocorticoid transport by P-glycoproteins depends upon the presence of an hydroxyl group at position 11 of corticosteroids and is enhanced by hydroxyl groups at the positions 16, 17, and 21. The antiprogestin RU486, which contains a dimethyl aminophenyl substitution at the position 11, is not transported by the mdr1 P-glycoprotein. We have found that RU486 is an inhibitor of P-glycoprotein function, indicating that steroid analogs could be useful chemosensitizers in patients undergoing chemotherapy.

Reversal of multidrug resistance by RU 486.

P-Glycoproteins represent a family of drug efflux proteins that convey multidrug resistance to cells in which they are expressed. This phenomenon can lower the efficacy of drugs used in chemotherapy. The steroid progesterone has been shown to bind P-glycoproteins and inhibit their drug efflux. We report that the antiprogestin RU 486 can reverse multidrug resistance in cells overexpressing the mouse mdr1 gene. Using flow cytometry to measure inhibition of P-glycoprotein-dependent efflux of rhodamine 123, RU 486 was found to be considerably more effective than progesterone and one-half as effective as verapamil. The results suggest a valuable new use for RU 486.

Expression of an mdr gene is associated with a new form of resistance to dexamethasone-induced apoptosis.

A variant, MS23, of murine thymoma W7 cells, previously selected for its resistance to low concentrations of dexamethasone, is cross-resistant to unrelated drugs such as puromycin and colchicine. We report here that transcription of the mouse mdr1 gene is activated and P-glycoprotein is expressed in MS23 cells. Moreover, additional variants with increased resistance to dexamethasone and other drugs can be isolated from MS23 by stepwise selections in dexamethasone and colchicine. In one such variant (S7CD-5), the mdr1 gene is amplified and the mdr protein overexpressed. These variants have classical mdr characteristics: they accumulate reduced concentrations of drugs (including dexamethasone), and both drug sensitivity and intracellular accumulation can be restored by verapamil. The variants are most resistant to glucocorticoids with both 11- and 17-hydroxyl groups. The results indicate that we have identified a new form of glucocorticoid resistance, one associated with expression of the mouse mdr1 P-glycoprotein.

Synergistic induction of apoptosis with glucocorticoids and 3',5'-cyclic adenosine monophosphate reveals agonist activity by RU 486.

Glucocorticoids induce a programmed cell death in immature murine T cells through a process that has been named apoptosis. Cyclic AMP-dependent protein kinase (PKA) activity contributes to this response but acts through an undefined mechanism. Steroid-induced cytolysis can be completely blocked by the glucocorticoid antagonist RU 486, which inhibits the transformation of the glucocorticoid receptor (GR) into a fully activated transcription factor. However, the ability of cAMP to act synergistically with steroids to cause apoptosis has revealed that a limited portion of RU 486-bound GR can translocate to the nucleus and contribute to a loss of cell viability. The combination of cAMP and RU 486 was also found to act cooperatively to regulate mRNA levels of specific genes. A 5-kilobase VL30 retroviral element transcript, which had previously been shown to be regulated synergistically by cAMP and dexamethasone, was strongly induced by the combination of RU 486 and cAMP. There was no agonist effect of RU 486 on the induction of the VL30 5-kilobase transcript in a variant cell line with defective PKA. Thus, the ability of RU 486 to act as an agonist, in this instance, was cAMP dependent. A similar response was also seen with the chondroitin sulfate proteoglycan core protein gene. On the other hand, mouse mammary tumor virus mRNA levels, which were not affected by cAMP alone, did not respond to a combination of RU 486 and cAMP. Studies of GR function, evaluating nuclear translocation and DNA binding at a GR-specific DNA regulatory element site found no evidence for a general effect of PKA activation on these receptor functions. We propose that cAMP may contribute to the induction of apoptosis at a step beyond receptor transformation by promoting an interaction between GR and other gene-specific regulatory proteins. Moreover, this interaction is sufficient to overcome the inhibition imposed by RU 486 on the functional capacity of nuclear glucocorticoid receptors.

Transformation of glucocorticoid receptors bound to the antagonist RU 486: effects of alkaline phosphatase.

RU 486 is a synthetic steroid that binds avidly to glucocorticoid receptors without promoting their transformation into activated transcription factors. A significant part of this behavior has been shown to be due to a failure of the RU 486 bound receptor to be efficiently released from a larger (sedimenting at 8-9 S) multimeric complex containing the 90-kDa heat shock protein. Our studies have found that in vitro at 15 degrees C the RU 486-receptor was slowly released from the 8-9S complex and converted into a DNA binding protein by a process that could be blocked by sodium fluoride. Moreover, this transition was significantly accelerated by treatment with alkaline phosphatase. High-resolution anion-exchange chromatography showed that the profile of receptor subspecies released from the 8-9S complex (in the absence of phosphatase treatment) was different for the RU 486 bound receptor when compared to the receptor occupied by the agonist triamcinolone acetonide. Production of the earliest eluting receptor form (peak A) was inhibited with RU 486. Peak A had previously been shown to be the predominant form of the receptor possessing a capacity to bind DNA. Treatment of the RU 486-receptor with alkaline phosphatase increased the formation of the peak A subspecies as well as the capacity of receptor to bind DNA-cellulose. Taken together, the results indicate that phosphorylation of the receptor or a tightly bound factor contributes to defining the capacity with which individual steroids can promote dissociation of the 8-9S complex and conversion of the glucocorticoid receptor into a DNA-binding protein.

Cyclic AMP-dependent protein kinase modulation of the glucocorticoid-induced cytolytic response in murine T-lymphoma cells.

The antiproliferative effect of glucocorticoid hormones on lymphoid tissue serves as the basis for their use in chemotherapy of lymphomas and leukemias. The effectiveness of the steroid-mediated response is potentially contingent upon a variety of factors, including the cellular level of glucocorticoid receptors. This report demonstrates that differences in the expression of the glucocorticoid receptor gene can modulate steroid sensitivity of individuals within a population of lymphoma cells. We have also found that loss of cAMP-dependent protein kinase activity caused a measurable decrease of steroid sensitivity in the murine T-lymphoma WEHI-7 without producing a significant change in steroid binding capacity. However, the extent of this change in sensitivity was dependent upon the level of glucocorticoid receptor expression. Lymphoma cells containing few spare steroid receptors became significantly resistant to glucocorticoids through loss of cAMP-dependent kinase function. On the other hand, elevated levels of cAMP were found to cause an increase in glucocorticoid receptor mRNA concentrations. Thus, cAMP-dependent protein kinase activity has the potential to modulate a lymphoma cell's steroid sensitivity by affecting the level of glucocorticoid receptor expression as well as the receptor's efficiency in producing a cytolytic response.

Transformed glucocorticoid receptors consist of multiple subspecies with differing capacities to bind DNA-cellulose.

The glucocorticoid receptor can be transformed into a DNA-binding protein by a process that is both hormone and temperature dependent. We have used a modification of the conventional method of anion-exchange chromatography to separate and analyze a variety of receptor subspecies that result from this transition. One receptor form (peak A) was found to have a capacity to bind DNA-cellulose which was significantly greater than that of the other species. Under conditions of mild heating (15 degrees C), the relative abundance of peak A in the receptor population and the rate of receptor transformation were both increased as a result of incubating samples with alkaline phosphatase. The mechanism appears to involve the conversion of the more "acidic" forms into that of peak A. The results indicate that receptor transformation is a multistep process which may be promoted by the removal of phosphate from either the receptor or a receptor-bound regulatory factor.

Analysis of glucocorticoid receptor subspecies binding to DNA-cellulose and isolated nuclei.

Conversion of the glucocorticoid receptor into a DNA-binding protein results in the generation of several distinct receptor subspecies (peaks A-E) which can be resolved by anion exchange chromatography. In vitro, the fraction of the receptor population (approx. 40%) which gains a capacity to bind DNA-cellulose is preferentially transformed into the peak A species by a process that was enhanced by the presence of KCl. At 0.4 M KCl, virtually all of the DNA-binding receptor was in the peak A form. Isolated nuclei also exhibit a receptor binding profile similar to that observed with DNA-cellulose.

High-resolution anion exchange chromatography of the glucocorticoid receptor from WEHI-7 cells.

A simple refinement of the current methods of DEAE chromatography of steroid receptors has been developed which takes advantage of the characteristics of Fast Flow DEAE Sepharose (Pharmacia). The approach provides a convenient and inexpensive means to carry out high-resolution chromatography of the glucocorticoid receptor. Using this method, at least five separate species of receptor have been detected within the single so-called "low-salt" peak normally seen using the current methods of receptor anion exchange chromatography.

The c-erb-A gene encodes a thyroid hormone receptor.

The cDNA sequence of human c-erb-A, the cellular counterpart of the viral oncogene v-erb-A, indicates that the protein encoded by the gene is related to the steroid hormone receptors. Binding studies with the protein show it to be a receptor for thyroid hormones.

Cyclic AMP-dependent protein kinase promotes glucocorticoid receptor function.

Murine lymphoma cell lines such as WEHI-7 exhibit a cytolytic response to both cAMP and glucocorticoids. We have exploited this behavior to ask if cyclic AMP-dependent protein kinase plays a role in regulating glucocorticoid receptor function. We have found that cAMP-resistant cell lines containing a defective cAMP-dependent protein kinase activity give rise to spontaneous steroid-resistant variants at a high frequency (approximately 10(-7)) relative to wild type cells (less than 10(-10)). Unlike previous results with wild type cells, nearly complete loss of glucocorticoid receptor function was observed in a single selection using unmutagenized cAMPr derivatives of WEHI-7. Thus, the initial selection of the cAMPr phenotype serves as a permissive step toward the acquisition of glucocorticoid resistance in WEHI-7. In addition, cAMP was found to increase the levels of steroid binding in these cell lines, and the dose response was dependent upon the phenotype of the cyclic AMP-dependent protein kinase. The results demonstrate an important role for cAMP in regulating glucocorticoid receptor activity and strongly suggest that this novel two-step selection scheme leads to the isolation of new forms of glucocorticoid resistance.

Isolation of new types of dexamethasone-resistant variants from a cAMP-resistant lymphoma.

We have developed a sequential selection procedure for the isolation of novel steroid-resistant variants of the murine thymoma WEHI-7. The first step involves the isolation of cell lines with an altered cAMP-dependent protein kinase (cAPK) activity by selection for resistance to dibutyryl cAMP (dbcAMP). The second step involves the selection for resistance to dexamethasone (dex) which results in the isolation of variants with decreased receptor function and a cAMPrdexr phenotype. The initial selection, to cAMPr, serves as a permissive step since isolation of spontaneous glucocorticoid resistance from wild-type WEHI-7 does not occur at a measurable frequency. The results demonstrate a potential role for cAPK in regulating the functional levels of glucocorticoid receptor and suggest that mutations in other cellular functions that affect receptor activity could lead to steroid resistance in lymphoid cells.

Characterization of the glucocorticoid receptor. Comparison of wild type and variant receptors.

We have measured the size of the glucocorticoid receptors from two murine lymphoid cell lines, one displaying a wild type cytolytic response to hormone, the other a resistant variant. Using radiation inactivation and target analysis, we first compared the nuclear and cytoplasmic forms of the steroid receptors in a wild type line, WEHI 7.1 (W7). Within the variation expected for this type of measurement (+/- 14%), the nuclear and cytoplasmic forms have the same size, 75,000 and 79,000 daltons, respectively. We have also measured the size of the receptor in a hormone-insensitive "nuclear transfer-increased" (nti) variant (S49 143R). In contrast to reports indicating that the nti phenotype is associated with a much smaller cytoplasmic receptor, we found little or no difference in sizes of translocated receptor in wild type and nti cells. We have found significant differences, however, in the release of wild type and nti receptors from nuclei by nuclease digestion, salt, and spermidine. Approximately 80% of the nti receptor was readily released from nuclei incubated with micrococcal nuclease, while only 40-50% of the wild type receptor was released under similar conditions. The wild type nuclei also contained a fraction of receptor (approximately 30%) which was resistant to extraction by NaCl and spermidine. This fraction was greatly diminished in the nti nuclei. Thus, a portion of the wild type receptors appears to be stabilized within the nuclei, possibly through a type of interaction which cannot be sustained by the nti receptor.

Phenothiazines cause a shift in the cAMP dose-response: selection of resistant variants in a murine thymoma line.

Cyclic AMP and glucocorticoid hormones each promote a cytolytic response in the murine lymphoid cell line WEHI-7.1 (W7). The sensitivity to dibutyryl cyclic AMP (dbcAMP), but not dexamethasone, can be enhanced by several psychotropic drugs that have the capacity to interfere with a variety of calcium-regulated functions. We have characterized the response of W7 cells to the phenothiazine trifluoperazine (TFP) and found that TFP concentrations, which decreased the growth rate by twofold, shifted the dose response to dbcAMP approximately tenfold. A similar but smaller shift was seen with the phosphodiesterase inhibitor methylisobutylxanthine (MIX). The effects of TFP and MIX on the dbcAMP response were additive, suggesting that TFP may act to increase the sensitivity of W7 cells to the action of cAMP-dependent protein kinase, and that the increased sensitivity may be due to altered lytic functions coregulated by both cAMP and calcium. The change in the dose response to dbcAMP caused by TFP provided a means of selection to obtain variants that were altered in their capacity to respond to dbcAMP, TFP, or the combination of the two drugs. Eight (out of 36) of the lines that were obtained after a mutagenesis and combined drug selection have been partially characterized. This has revealed the existence of several new phenotypes. Most have an altered response to dbcAMP in the presence of TFP and are likely to represent variants that have not been observed before.

Nuclear interactions of wild type and variant glucocorticoid receptors.

Nuclease digestion of nuclei from glucocorticoid sensitive and resistant lymphoma cell lines was used to study the nuclear compartmentalization of wild type and variant glucocorticoid receptors. In comparison with wild type, the variant line (S49 143r) had an increased capacity to translocate to the nucleus (nti), but was more completely released from nuclei by nuclease digestion. Approximately 20% of the receptor in wild type nuclei was resistant to release by DNase I digestion, while only less than 5% of the receptor from nti nuclei was retained under the same conditions. Studies with wild type nuclei show that the nuclease resistant portion of receptors was also more resistant to release by increased ionic strength.

The size of the thyroid hormone receptor in chromatin.

We have used radiation inactivation and target theory to determine the size of the functional unit for T3 binding in rat liver chromatin. The process involves exposure of frozen chromatin samples to a beam of high energy electrons produced in a linear accelerator and subsequent measurement of the residual capacity to bind hormone. Our experiments were carried out using three forms of solubilized chromatin: 1) sonicated, containing the receptor in fragments which sedimented faster than 30 S; 2) digested by nuclease, containing the receptor in a form which sedimented at 5-6 S; 3) digested by nuclease and made 0.5 M in KCl, containing the receptor in a form which sedimented at 3.8 S. We have shown that in each sample preparation the receptor retained the ability to bind T3 with the same capacity and affinity that had previously been measured with high molecular weight chromatin. Irradiation caused a reduction in the capacity to bind T3 but did not change the affinity of the remaining receptors for the hormone. In each preparation, the radiation resulted in a simple exponential loss of binding capacity with dose, indicating that a single target size was detected. Within the variation of the measurements, the target size for each form of the receptor was the same, 59,000 daltons.