Induction of Parathyroid Hormone

Home , David Goltzman, Tetrapeptide

(CANCER RESEARCH 57. 4517-4522. October 15. I9>)7| Induction of Parathyroid Hormone-related Peptide by the Ras Oncogene: Role of Ras Farnesylation Inhibitors as Potential Therapeutic Agents for Hypercalcemia of Malignancy1

Fasika Aklilu, Morag Park, David Goltzman, and Shafaat Ahmed Rabbani2

Department of Medicine, McGill University and Calcium Research Laboratory and Molecular Oncology Group. Royal Victoria Hospital. Montreal. Quebec, H3A IAI Canada

ABSTRACT into the circulation and acts at the level of bone to stimulate bone rÃ©sorptionand Ca2^ release and on the kidneys to increase Ca2 + Parathyroid hormone related peptide (PTHRP) is the major causal reabsorption (19). The outcome of these actions is the development of agent in the syndrome of malignancy-associated hypercalcemia (MAH). hypercalcemia, which may be fatal. Several studies have shown that PTHRP production is increased in re sponse to growth factors and oncogenes, such as Tpr-Met, that are asso Increasing number of studies on the regulation of PTHRP gene ciated with the tyrosine kinase signaling pathway. Using site-directed expression are beginning to shed light on the molecular mechanisms mutagenesis of Tpr-Met and chemical inhibitors of phosphotidylinositol-3 responsible for PTHRP overproduction by cancer (20, 21 ). We have kinase and Ras isoprenylation, we demonstrated previously that induction previously shown the critical role played by growth factors, acting on of PTHRP is mediated via the Ras signaling pathway. In the present study, tyrosine kinase receptors, in stimulating PTHRP production (22). We we have directly investigated the role of the Ras oncogene in MAH. As a subsequently demonstrated the effect of the oncogenic derivative of model system, we used Fisher rat 3T3 fibroblasts stably transfected with the hepatocyte growth factor/scatter factor receptor, Tpr-Met, on a Ras oncogene (Ras-3T3). Ras transfection enhanced PTHRP production increasing PTHRP production and demonstrated that the mechanism 5-10-fold in these cells, and inoculation of this cell line into nude mice led of this stimulation involves the intracellular Ras signaling pathway to the development of hypercalcemia within 2 weeks. We used this system (22). Previous studies by us and others, examining the relationship to evaluate the effect of a potent inhibitor of Ras processing, B-1086, on between tyrosine kinases and PTHRP, have indeed indicated that Ras cell growth, PTHRP production, plasma calcium, and tumor growth. Treatment of Ras-3T3 cells in vitro with B-1086 at 0.1-10 Â¿ig/mlproduced is at the cross-roads through which the signal for PTHRP induction is a significant reduction in PTHRP mRNA expression and PTHRP secre propagated. tion and a significant decrease in cell proliferation. Treatment in vivo of Ras genes play a fundamental role in basic cellular functions (23). BALB/c/nu/nu mice bearing Ras-3T3 tumors with B-1086 resulted in a Ras proteins are GDP/GTP-regulated switches that in their mature significant inhibition in tumor growth. In addition, this treatment pro functional form are anchored to the cell membrane by an isoprenyl tail duced near normalization of serum C'a2*, a significant decrease in plasma (24, 25). In normal physiology. Ras functions in transducing devel PTHRP, and a reduction in tumoral PTHRP mRNA levels. These results opmental and proliferative signals from the cell surface to the nucleus. show that the Ras pathway is involved in PTHRP production by tumors, In addition to its role in growth and differentiation. Ras is also identifies Ras as a potential target for treatment of MAH, and demon important in controlling cytoskeletal protein expression and in regu strates Ras processing inhibitors as candidate therapeutic agents against lating vesicle trafficking within the cell (26). Perhaps owing to its this syndrome. central role within the cell, alteration of this gene is often associated with the initiating steps that lead toward carcinogenesis. Indeed, INTRODUCTION activating mutations in the Rax gene account for at least 40% of Hypercalcemia of malignancy is a major complication in a signif human neoplasia and play a significant role in progression of these icant portion of cancer patients and PTHRP1 has been identified as the malignancies (27). In light of this, Ras is an ideal target for therapeutic major pathogenic factor for this disease (1-5). PTHRP shares a high intervention to combat both the establishment of the primary cancer degree of homology in its NHrterminal region with PTH and because and for the prevention of secondary syndromes (28). One approach to of this sequence homology is able to mimic the actions of PTH by target Ras has been the development of peptidomimetic inhibitors of interacting with a common PTH/PTHRP receptor (6. 7). In normal Ras, which mimic the farnesylation site of the Ras protein, a CAAX physiology, PTHRP is widely expressed, acts in an autocrine or consensus sequence at the COOH-terminal, and act by competitively paracrine manner, and has been shown to have a broad spectrum of inhibiting Ras farnesylation (29, 30). Farnesylation is essential for functions especially related to modulating cell growth and differenti subsequent modifications of Ras, for membrane localization, and ation (8-11 ). PTHRP appears to be a key regulator of skeletal devel ultimately for functioning of this oncogenic protein (31). opment in the fetus but has also been implicated in transepithelial In the present study, using Fisher rat 3T3 fibroblasts transfected calcium transport in such sites as mammary epithelium, kidney, and with the Ras oncogene, we have examined the role of Ras in PTHRP placenta (12-16). Furthermore, PTHRP has been shown to be a potent production and in the development of hypercalcemia in vivo. We have muscle relaxant acting on the smooth muscle of the uterus, urinary also tested the ability of a Ras farnesylation inhibitor B-1086 to alter bladder, and vascular wall (17). In malignancy-associated hypercal the course of the tumor and its production of PTHRP and assessed the cemia, PTHRP acts primarily as an endocrine factor (18). When large effects on the hypercalcÃ©miesyndrome in vivo. quantities of PTHRP are produced by cancer cells, PTHRP is secreted

MATERIALS AND METHODS Received 5/29/97; accepted 8/18/97. The costs of publication of this article were defrayed in part by the payment of page Ras Farnesylation Inhibitor B-1086. The Ras farnesyl transterase inhib charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. itor. B-1086 (29). was kindly provided by Dr. Michael Lewis of Eisai Research 1This work was supported by Medical Research Council of Canada Grants MT-10630. Institute (Andover. MA). MT-12609. and MT-5775 and by a grant from the National Cancer Institute of Canada. 2 To whom requests for reprints should be addressed, at Calcium Research Laboratory. Animal Protocols. Female BALB/c/iui/nu mice (Charles River Breeding Laboratories, Wilmington. MA), weighing 15-20 g (4-5 weeks of age) were Room H4-72. Royal Victoria Hospital. 687 Pine Avenue West. Montreal. Quebec. H3A injected on day 0 s.c. into the left and right flanks with Ras-3T3 cells ( 1 X IO5) 1A1 Canada. Phone: (5141843-1632; Fax: (514)843-1712. 1The abbreviation used is: PTHRP. parathyroid hormone-related peptide. suspended in 0.1 ml of sterile saline. Animals were housed individually and fed 4517

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1997 American Association for Cancer Research. INDUCTION OF PTHRP BY THK A,i.i ONCOGENE ad lihitiim on autoclaved standard rodent chow (rat chow 5012: Ralston-Purina ':P|dCTP as described previously (22. 33). After a 24-h incubation at 42Â°C, Canada. Inc.. Lasalle. Quebec. Canada) containing \r/c calcium and 0.74% filters were washed twice under low stringency conditions ( 1X SSC and phosphorus and sterilized tap water (32). On day 5, vehicle. 50 mg/kg B-1086. 1% SDS: at room temperature for 40 min) and under high stringency or 100 mg/kg B-1086 was administered daily i.p. into anaesthetized animals. conditions (0.1 X SSC, 0.1% SDS; at 55Â°Cfor 40 min). Autoradiography of All animals were examined for the development of tumors daily for up to 15 filters was carried out at â€”¿70Â°CusingXAR film (Eastman Kodak Co., days. On the day of sacrifice, animals were bled by intracardiac puncture, Rochester. NY) with two intensifying screens or by using a phosphorim- and tumor volume of control and experimental animals was measured in ager screen (Eastman Kodak Co.. Rochester. NY). The level of PTHRP two dimensions by calipers, and the tumor volume was calculated expression was quantified by densitometric scanning using the Mac BAS ((width + length/2)1]. On the day of sacrifice, animals were bled by intracar VI.01 alias program. diac puncture, and tumor tissue was extracted and immediately frozen in an Ras Processing Assay. Ras-3T3 cells were plated in medium containing ethanol bath for RNA analysis. 10% calf serum, and 24 h later, treated with 0. 10, or 50 Â¿ig/mlof B-1086 for Cells and Cell Growth. The Fisher rat (Fr) 3T3 fibroblasts expressing the 72 h and harvested in lysis buffer (PBS. 1% NP40, 0.5% sodium deoxycholate. Ras oncogene were generated by ecotropic retroviral infection as described 0.1% SDS, 10 ptl/ml PMSF. 30 /Ml/ml aprotinin, and 10 /il/ml sodium or- previously (22). Transient transfection into COS-1 cells of the pLXSN retro- thovanadate). The lysates (25 fj.g) were electrophoresed on a 15% SDS- viral vector encoding the Kux gene and G418 resistance and of the pSV-Y-E- polyacrylamide gel. transferred to nitrocellulose membranes, and immuno- MLV plasmid encoding retroviral packaging proteins was performed by the blotted with anti-ras antibody (Santa Cruz Biotechnology). The signals were DEAE-dextran method. At 12 h after transfection, cells were treated with 100 visualized using chemiluminescence (Amersham Corp.). mM chloroquine for 3 h and incubated for an additional 48-72 h before harvesting. Transfected COS-1 cells were lysed. and the supernatant contain Other Analytical Methods. For RIA. conditioned medium (2 ml/well of a ing the packaging virus, al a titer of 5 X 10' plaque-forming units, was used six-well cluster plate) was removed from control and experimental 3T3 cells treated with vehicle alone or with graded doses of B-1086 for 24 h. Duplicate to infect Fr 3T3 cells. Experimental cell lines were then selected for G4I8 2(X)-500-(j.l aliquots of cell-conditioned culture medium were evaporated to resistance and foci formation. dryness in a Speedvac (Savant Instruments. Inc.. Hicksville. NY) and stored at For growth curves. Fr 3T3 cells were plated in medium containing 2% fetal â€”¿20Â°Cuntil assayed. Dried medium was reconstituted with 200 /j.1of outdated bovine serum in 9.6-cnr Petri dishes (Falcon) at seeding densities of 5 X IO3 cells/plate. Cells from replicate dishes were trypsinized and counted, daily, in blood bank plasma and radioimmunoassayed as described previously using 125l-labeled [Tyr] PTHRP(l-34) as a tracer and PTHRP(l-34) as a standard a model Z Coulter counter (Coulter Electronics. Hialeah, FL). Medium was (34). Results are expressed as nanogram equivalents of PTHRP(l-34)/101' changed in all plates every 24 h (22). Northern Blot Analysis. Total cellular RNA was isolated from the cells. control and experimental cells, following treatment with vehicle alone or Serum calcium levels were determined by atomic absorption spectropho- graded concentration of B-1086, by acid guanidinium thiocyanate-phenol- tometry (model 703; Perkin-Elmer, Norwalk. CT). and plasma PTHRP was chloroform extraction as described previously (22). Twenty /ng of total assayed using a commercial two-site immunoradiometric assay IRMA cellular RNA were electrophoresed on a 1.1% agarose-formaldehyde gel. (Nichols Institute Diagnostics. San Juan Capistrano. CA). transferred to a nylon membrane (Nytran) by capillary blotting, and then Statistical Analysis. Results are expressed as the mean Â±SE of at least fixed by drying and UV cross-linking for IO min. The integrity of the RNA triplicate determinations, and statistical comparisons are based on the Student's was assessed by ethidium bromide staining. Hybridization was carried out t test or ANOVA (35). A probability value of <0.05 was considered to be with "P-labeled PTHRP cDNA and with an 18S RNA probe using |a- significant.

A. B.

n > OC

PTHRP Fig. 1. Effect of Ras expression on PTHRP pro duction. A. Northern blot analysis of Fr 3T3 cells infected with empty vector alone (V) or with exper imental vector

â€¢¿50.5-

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comparison was made with the amount of PTHRP released into the B-1086 wild-type and vector-transfected 3T3 cells. Secretion of immunoreactive PTHRP was significantly greater in 3T3 cells transfected with Ras as compared to control cells (Fig. IÃŸ).These results demonstrated that Ras itself was a potent inducer of PTHRP production, independ ent of any growth factors present in the cell culture medium. Effect of IM 086 on Ras Processing and PTHRP Production by 10 50 Ras-3T3 Cells in Vitro. To examine the capacity of B-1086 to inhibit Ras processing. Ras 3T3 cells were treated with B-1086 (10 or 50 jag/ml). B-1086 produced a dose-dependent reduction in Ras protein processing in Ras-3T3 cells (Fig. 2). Treatment with vehicle alone (0.1% DMSO) revealed no inhibitory effect on Ras processing. The ability of B-1086 to influence PTHRP production was exam ined in experimental Ras-3T3 cells in vitro. Ras-3T3 cells were treated with increasing concentrations (0.01-10 /ng/ml) of B-1086 for Ras-3T3 48 h. At the end of this treatment, cells were isolated, and total cellular Fig. 2. Treatment with B-1086 inhibits Ras protein processing in Ras-3T3 cells in vitro. RNA was extracted and analyzed for PTHRP mRNA expression by Ras-3T3 cells were grown to 70% confluence and treated with either vehicle. 10 Â¿ig/mlof Northern blot analysis. Conditioned medium from control and exper B-I086. or 50 (ig/ml of B-I086 for 72 h. The cell lysates were electrophoresed and immunohlotted with anti-Ras antibody and analyzed for Ras processing as described in imental cells was assayed for immunoreactive PTHRP by an NH,- "Materials and Methods". U. unprocessed; P. processed forms of Ras. terminal PTHRP RIA. B-1086 caused a dose-dependent decrease in PTHRP mRNA expression (Fig. 3/1) and secretion of PTHRP protein into conditioned culture medium (Fig. 3ÃŸ).Morphological analysis of RESULTS Ras-3T3 cells following treatment with graded doses of B-1086 and Effect of the Ras Oncogene on PTHRP Production. To examine cell viability assay using trypan blue failed to show any cytotoxic the effect of Ras oncogene transfection on PTHRP production, total effects of B-1086 in concentrations used in this study (data not cellular RNA was extracted from control and /fo.v-transfected cells shown). and analyzed by Northern blot analysis. Both control and experimen Effect of B-1086 on the Growth of Ras-3T3 Cells. The effect of tal cell lines were maintained in culture for at least 24 h in the absence cell growth following transfection with the Ras oncogene was exam of serum to rule out any change in PTHRP expression by serum- ined for 6 days in viiro, and comparison was made with the growth of derived growth factors. Transfection of the Ras oncogene caused a wild-type cells and with Ras-3T3 cells treated with B-1086 (10 marked increase (5-10-fold) in PTHRP mRNA expression (Fig. U). Â¿ig/ml).Transfection of the Ras oncogene caused a significant in The effect of transfection of this oncogene on PTHRP secretion into crease in cell growth as compared to wild-type cells or cells trans the cell-conditioned culture medium was determined by RIA, and fected with vector alone (data not shown). Treatment of Ras-3T3 cells

B-1086(i,gml) B.

0 0.1 1.0 10

PTHRP

Fig. 3. Effect of B-1086 on PTHRP production in Ras-3T3 cells. A. Fr 3T3 cells transfected with 18S the empty vector (V] or with the experimental plasmid (Rasi were grown to 70% confluence. Ras- transfected cells were incubated with 0. 0.1, or 10 /ig/ml B-1086. Twenty (ig of total cellular RNA were extracted from control and experimental cells in 2.5 -, and electrophoresed on a 1.1% agarose formalde hyde gel. Filters were probed with a 12P-labeled PTHRP cDNA or with a 32P-labeled 18 S RNA oc probe as described in "Materials and Methods." All blots were quantified by densitometric scanning (lower panel). B. conditioned culture medium from i control Fr 3T3 cells (V) and from /?Â«.v-transferredFr o. 3T3 cells treated with graded concentrations of K B-1086 was collected after 48 h and analyzed for PTHRP immunoreactivity as described in "Materi als and Methods." Significant differences from con 'SI trol cells are represented by asterisks. Bars, SE. *. P < 0.05. o I

V 0 0.1 1.0 10 V 0 0.1 1.0 10 B-1086(,Â¡g ml) B-1086 (ng/ml)

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trast to PTH, the secretion of which is tightly regulated by concen trations of the extracellular calcium ion such that calcimimetics acting on the calcium receptor can block PTH secretion, it appeared unlikely that attempts to reduce PTHRP release by inhibition of secretion would be rewarding (36). Indeed, the rate of PTHRP secretion appears to be a direct function of its rate of synthesis, and the capacity of a given tumor to release PTHRP has been found to correlate with its level of gene expression (37, 38). Consequently, we have used alter native strategies to inhibit PTH release in vitro and to test these effects on in vivo models. One approach we used previously emanated from our in vitro observations that PTHRP gene transcription could be inhibited by the active metabolite of vitamin D, 1,25-dihydroxyvitamin D (11, 39). Indeed, 1,25-dihydroxyvitamin D and a nonhypercalcemic analogue were then shown to diminish PTHRP production by tumors in vivo (40). A second approach we have used was to employ antisense technology to diminish PTHRP translation in vitro, and this strategy Time (days) also reduced PTHRP production by tumors in vivo (41). Finally, a third strategy was to inhibit the postranslational processing of PTHRP. Fig. 4. Effecl of B-1086 on growth curves of Ras-3T3 cells. Growth curves of wild-type cells (A) or /?Â«.v-transfected3T3 cells either untreated (O) or treated (â€¢)with PTHRP is synthesized as a prohormone, which has low intrinsic 10 Â¿Â¿g/mlB1086. Cultures were seeded at a density of 5.0 X 104 cells/dish and incubated bioactivity; when the prohormone sequence is removed, the mature for the indicated periods of time in standard culture medium. Cells from triplicate dishes were trypsinized and counted as described in "Materials and Methods." Each point form is a fully active entity. We demonstrated that this cleavage can represents the mean of four different experiments; bars. SE. Significant differences from be accomplished by a furin-like prohormone convertase (42) and then Ras-3T3 cells treated with vehicle alone are represented by asterisks. *, P < 0.05. showed that inhibition of the action of furin in tumors that overpro duce PTHRP reduces levels of bioactive PTHRP in vitro and is also effective in vivo (41, 42). Consequently, several approaches may be with B-1086 caused a marked decrease in cell doubling time and used based on mechanisms of PTHRP regulation that have been growth was reduced to a rate comparable with that seen in untrans- determined in vitro, which can diminish PTHRP production by tumors fected 3T3 cells (Fig. 4). implanted in vivo. Effect of Infusion of B-10S6 on Tumor Volume in Vivo. BALE/ The use of Ras farnesylation inhibitors to inhibit PTHRP produc clnu/nu mice inoculated with experimental Ras-3T3 cells developed tion was based on our initial observations that growth factors such as palpable tumors by 5 days after tumor cell inoculation. These tumors epidermal growth factor or insulin-like growth factor I can be potent continued to increase in volume for the next 10 days. Experimental stimulators of PTHRP gene transcription in vitro (11, 14, 37). Sub animals were inoculated with Ras-3T3, and beginning on day 5 after sequently, we reported that an oncogenic, constitutively active, tumor inoculation, animals were injected daily with either vehicle growth factor receptor, Tpr-Met, could also increase PTHRP produc- alone or with different concentrations (50 or 100 mg/kg) of B-1086. Animals infused with B-1086 showed a marked decrease in tumor growth as compared to animals infused with vehicle alone, and the effects of B-1086 on decreasing tumor volume was dose dependent (Fig. 5). 20-, IIIIIIIIII Effect of B-1086 on Serum Ca2+ of Ras-3T3 Tumor-bearing Mice. Ras-3T3 cells were inoculated into the right flank of nude mice, and animals were monitored for the development of hypercalcemia. Inoculation of Ras-3T3 cells resulted in the development of palpable tumors by 6 days and hypercalcemia by day 15 after tumor cell inoculation. Treatment of these experimental animals with B- 1086 (50 or 100 mg/kg) for 10 days (day 6-15) resulted in a signif icant reduction in their serum Ca2+ levels, which appeared to be dose dependent (Fig. 6). Effect of B-1086 on Tumoral PTHRP Production in Vivo. On day 15 after tumor cell inoculation, all control and experimental animals were sacrificed, and their tumors were removed to analyze the level of tumoral PTHRP mRNA expression. Treatment of experimen tal animals with B-1086 (100 mg/kg) caused a significant decrease in PTHRP mRNA expression (Fig. 7/4). Also at the time of sacrifice, on day 15, blood was collected and analyzed for plasma PTHRP. Treat ment of tumor-bearing mice with B-1086 caused a significant de Time (days) crease in their plasma PTHRP levels (Fig. IB). Fig. 5. Effect of B-1086 on the tumor volume of Ras-3T3 tumor-bearing animals. Tumor-bearing animals were injected daily with 50 mg/kg (â€¢)or 100 mg/kg (A) of B-1086, and tumor volume was determined at daily intervals (arrows) as described in DISCUSSION "Materials and Methods." Comparison was made with tumor volume of animals receiving vehicle alone (untreated; A). Results represent the means of four starting animals in each In previous studies, we demonstrated that PTHRP secretion by a group in three different experiments; bars, SE. Significant differences from control tumor cell model is largely constitutive (32). Consequently, in con- tumor-bearing animals at each time point are represented by asterisks. *. P < 0.05. 4520

3.3 n growth factor to their receptors, activation of Ras proteins by ex change of bound GDP for GTP, and the triggering of a phosphoryl- ation cascade that involves raf kinase and mitogen-activated protein 3.1 kinase; the end result is the activation of nuclear transcription factors that can apparently enhance PTHRP gene transcription and that can also regulate cell growth (43-45). Ã‹ 29 Signal transduction by Ras is dependent on its capacity to localize to the plasma membrane which, in turn, is dependent on farnesylation u 2.7 of the cysteine in the CAAX consensus sequence at the COOH 3 terminus of the Ras protein (31 ). Inhibition of farnesylation is, there fore, being explored by several laboratories as a potential mechanism 2.5 for inhibiting tumor growth. In the present study, we used a small CD m organic molecule that mimics the tetrapeptide farnesylation site of Ras 2.3 protein but is a methyl ester prodrug. It is more potent than the free acid form of the drug (B956) in vitro, because of its higher membrane 0 permeability, but in vivo is rapidly hydrolyzed to the acid within the N CTL 50 100 circulation (29). This inhibitor, in common with other farnesyl trans- B-1086 ferase inhibitors (29-31), reduced Ras posttranslational processing Fig. 6. Effect of B-I086 on scrum calcium of Ras-3T3 tumor-hearing animals. and also decreased cell growth. Furthermore, infusion of different Tumor-hearing animals were infused with 50 or KM) mg/kg of B-1086 or vehicle (CTL) concentrations of B-1086 used in this study failed to show any and were sacrificed al the end of the experiment on day 15. Their serum calcium was then determined as described in "Materials and Methods." Serum calcium in non-tumor- evidence of toxicity or change in the body weight of experimental hearing animals is also shown (N). Results represent the means of at least three animals animals as compared to control animals receiving vehicle alone (29). in each group in three different experiments: bur*. SE. * and **. significant differences in The Rax oncogene is the site of one of the most prevalent genetic- serum calcium from normal animals IN) and from tumor-hearing control (CTL). respec tively (P < 0.05). alterations in human cancer and can occur in a broad spectrum of cancers, many of which have been demonstrated to overproduce PTHRP (20, 21). Consequently, inhibition of Ras action appeared to tion and that the Ras signaling pathway was involved in this stimu be a particularly effective site to target to inhibit PTHRP production lation. In the present study, we have shown that the Rax oncogene and the hypercalcÃ©miesyndrome as well as to reduce tumor growth. itself is a potent stimulus for PTHRP production in vitro and that Nevertheless, human cancers evolve as a result of multiple genetic overexpression of Ras in tumors in vivo can also lead to augmented alterations, and inhibition of more than one disordered function may PTHRP release and to the development of hypercalcemia. Further be needed to retard tumor progression. Therefore, with increased more, a potent inhibitor of Ras action can reverse these effects both in knowledge of the control of PTHRP production, targeting the multiple vitro and in vivo. sites of aberrant regulation may be the most effective means of The cascade leading to increased PTHRP production involves bind reversing increased secretion of this protein. Inhibition of Ras action ing of growth factors such as epidermal growth factor or hepatocyte should, nevertheless, be an important component of this approach.

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PTHRP Fig. 7. Effect of B-1086 on tumoral PTHRP production in tumor hearing animals. A. PTHRP (N mRNA expression was determined in tumor extracts as described Â¡n"Materials and Methods." PTHRP 18S mRNA expression after treatment with B-1086 ( I(X) mg/kg) was compared with PTHRP mRNA levels in control animals (CfLi .eceiving vehicle alone Imp panel). All blots were pruned with an I8S RNA cDNA as a control for RNA loading, and the den- 1.5 -i silometric ratios of PTHRP: 18S mRNA are shown (lower panel). B. PTHRP concentration was deter mined as described in "Materials and Methods" and OC I is expressed as pM PTHRP. Plasma PTHRP levels were examined in Ras-3T3 tumor-hearing animals <Ã» 1 - co receiving vehicle alone (CTL) or 100 mg/kg of f B-1086. Plasma PTHRP Â¡nnon-tumor-bearing ani D. CL E mals is also shown (AO. Results represent the means CE of three animals in each group in three different X experiments; bars. SE. D. detection limit of the * assay. Significant difference from control tumor- 5 0-5H bearing animals is represented by an asterisk. *. /' â€¢¿0.05. o i

CTL B-1086 N CTL B-1086

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1997 American Association for Cancer Research. Induction of Parathyroid Hormone-related Peptide by the Ras Oncogene: Role of Ras Farnesylation Inhibitors as Potential Therapeutic Agents for Hypercalcemia of Malignancy

Fasika Aklilu, Morag Park, David Goltzman, et al.

Cancer Res 1997;57:4517-4522.

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