- Email: [email protected]
Altered drug distribution and elevated expression of a novel transporter gene (MRP) in a multidrug resistant lung cancer cell line
4th LTBW Abstracts/Lung
350 lines in athymic nude mice. Whereas cell lines derived from tumors with a non-small cell lung carcinoma-like phenotype were significantly hypomethylated relative to their parental cell lines, a cell line derived from a tumor lung carcinoma-like phenotype retained of its parental cell line. The data indicate tion of normally unmethylated CpG
with a more small cell the methylation status that de novo methylaisland sequences and
phenotype-associated demethylation of nonisland sequences accompany immortalization and oncogene-induced transformation of bronchial epithelial cells. Furthermore, at each locus tested, these altered methylation patterns occur at different stages during this progression. Because the in vitro model indicated a particular sensitivity of bronchial epithelial cells to methylation at chromosome 17~13.3, we next examined the status of the pYNZ22 locus in primary tissue. As expected for a CpG island, the pYNZ22 region was found unmethylated in normal whole lung tissue. However, vitro studies showed the pYNZ22 locus to be partially ated in cultured NHBE. To determine whether the
to be our in methylpartial
methylation in NHBE cells was induced by cell culture or preexisted in the primary epithelium, DNA from isolated bronchial epithelium and the underlying stromal layer was examined. Whereas the epithelial cell DNA was partially methylated at this locus, DNA from the stromal layer was unmethylated, indicating that the partial methylation of pYNZ22 was specific to the epithelial cells. This partial methylation also appeared to be a specific feature of bronchial epithelium since DNA from neither primary colonic epithelium nor colonic stroma was methylated at this locus. Studies addressing the possibilities that partial methylation of the pYNZ22 locus in bronchial epithelium might predispose the region to further methylation during lung tumorigenesis or that methylation specifically related to bronchial epithelial underway.
of this locus is development are
~53 and prognosis Carbone DP”, Mitsudomi Tb, Rusch Vc, Nowak JAd, Mcintire D”, Chiba lb. Gazdar AF”, Minna JD”, The Lung Cancer Study Group. “Simmons Cancer Center, UT Southwestern, Dallas, TX, ‘NCI-NMOB, Bethesda, MD, “Memorial Sloan Kettering Cancer Center, NY, NY, and ‘Illinois Masonic Medical Center, Chicago, IL. Abnormalities in the gene structure, protein structure, expression level, and other properties of p.53 have been found to be common in a wide range of human tumors. Many investigators have established that small cell lung cancers have mutation rates that approach 100X, and ~53 mutations are found in about 50% of non-small cell lung cancers (NSCLC). Such common abnormalities indicate significance in the molecular pathogenesis of cancer, but what is the clinical significance of these findings? The results are mixed. Some groups have reported association of p.53 mutations with worsened survival only in early stage patients, others only in late stage patients. Some groups have reported association of immunostaining positivity with adverse outcome, and others find no such association. Many of these studies are hampered by small numbers of patients, short followup, incomplete staging, hap-
Cancer 10 (1994) 347-373
hazard patient accrual, different techniques of analysis, ferent treatment strategies at different institutions.
and dif-
In our own series, we investigated the association of ~53 abnormalities and survival in 85 patients with NSCLC resected with curative intent in 1987 and 1988 as part of the Lung Cancer Study Group protocol 871. These patients were all systematically staged and their slides reviewed by reference pathologists. Our previous studies showed that only a subset of ~53 mutations in lung cancers result in overexpression and protein overexpression has been described in the absence of mutation. Therefore, we determined both protein overexpression (by immunostaining) and gene mutations (by single strand conformation polymorphism and DNA sequencing) in these tumors. Clinical followup data was available for 74 cases (median followup of 1564 days). Of the showed ~53 overexpression and 54% quences; however the concordance rate was a strong negative survival correlation
studied patients 55% had mutant ~53 sewas only 65%. There with positive ~53 im-
munostaining (P = O.Ol), but not with the presence of gene mutations (P = 0.96) in this group of patients. Why might immunopositivity be more significant than genetic mutations? Overexpression of ~53 may independently contribute to adverse outcome due to the ability of ~53 to act as a dominant oncogene (positive or negative), or alternatively, overexpression may reflect a response to ongoing DNA damage in the tumor, which in turn may be a marker for a more aggressive behavior. Thus in our series we find, in a single group of patients with over 4 years of follow-up, that ~53 overexpression, but not ~53 gene mutation, is a negative prognostic factor for patients with resected NSCLC. In summary, the disparity of results reported by different groups may reflect the fact that if such associations exist, they are relatively weak, or show up only with long follow-up. How could such a common lesion not have clinical relevance? One explanation may be that ~53 is only one component in a pathway which is universally abnormal in lung cancer, and when ~53 is normal, lesions in other as yet unknown genes may block its function. All lung cancers would thus be functionally equivalent with respect to this pathway and their behavior would be independent of the presence or absence of ~53 mutations. Even the finding of strong negative prognostic factors in lung cancer, however, may not allow us to translate these tindings into significant future improvements in survival with the ineffective or marginally effective adjuvant therapies currently available. Altered
drug distribution
and elevated expression of a novel
transporter gene (MRP) in a multidrug resistant lung cancer cell line
Cole SPC, Mirski
SEL, Bhardwaj
G, Almquist
KC, Deeley,
RG. Cancer Research Laboratories. Queen’s University. Kingston, Ontario K7L 3N6, Canada. Despite the widespread occurrence of resistance to chemotherapeutic agents in both small cell and non-small cell lung cancers, the clinically relevant molecular mechanisms responsible for this resistance remain largely unknown The role of P-glycoprotein, the I ‘IO-kDa plasma membrane protein encod-
4th LTBW Abstracts/
Lung Cancer IO (1994) 347-373
ed by the MDRl gene that is usually associated with multidrug resistance, appears limited in this tumour type. Our laboratory investigations have focused on a model system consisting of three cell lines: the NCI-H69 (H69) SCLC cell line; a doxorubicin-selected multidrug resistant variant, H69AR, that does not overexpress P-glycoprotein [1,2]; and a drug-sensitive revertant cell line, H69PR [3]. Laser-assisted confocal fluorescence microscopy studies show that daunomycin is predominantly localized in the nucleus and nuclear membrane of H69 cells; in contrast, the drug is sequestered in vesicle-like extranuclear structures in H69AR cells. These observations suggest that altered subcellular drug distribution may play a role in the resistance of the H69AR cell line. By screening an H69AR cDNA library by differential hybridization, we have recently identified a 65kb mRNA that is overexpressed approximately IOO-fold in H69AR cells compared to H69 and H69PR cells [4]. This mRNA is also expressed in five of five NSCLC cell lines tested. Southern blotting and cytogenetic studies indicate that the major mechanism underlying the elevated expression of this mRNA in H69AR cells is gene amplification. The 6%kb mRNA encodes a 1531 amino acid protein we have designated ‘multidrug resistance-associated protein’ (MRP). MRP is a novel member of the ATP-binding cassette (ABC) superfamily of transport systems. Other human proteins belonging to this family include the MHC class II-linked peptide transporters (TAP1 and TAP2), the cystic fibrosis transmembrane conductance regulator (CFTR) and the P-glycoproteins (MDRI and MDR3). However, the protein that is most closely related to MRP is ItpgpA, a gene which confers resistance to arsenite and trivalent antimonials in Leishmania farentolae. Increased expression of a 180- to I90-kDa 100000 x g membraneassociated protein is detected in H69AR cells using antibodies raised against several synthetic MRP peptides. The apparent molecular mass of this protein is about 20 kDa greater than Pglycoprotein and it is N-glycosylated. Photoaffinity labeling experiments with [32P]azido-ATP shows that it binds ATP. The isolation and characterization of MRP along with its increased expression in association with altered subcellular drug distribution provides the tirst definitive molecular evidence for the existence of a transporter that may confer or at least contribute to multidrug resistance in lung cancer cell lines. However, insight into the mechanism of the involvement of MRP in drug resistance awaits knowledge of the subcellular localization of the protein and the nature of the molecules it transports. Furthermore, a role for MRP in clinical drug resistance remains to be established. (Supported by MRC, NCIC and Cancer Research Sot.; SPCC is a Career Scientist of the OCTRF and RGD is the Queens University Stauffer Research Professor of Cancer Research.)
1 2 3 4
Mirski SEL, Gerlach JH, Cole SPC. Cancer Res 1987; 47: 2594. Cole SPC, Chanda ER, Dicke FP, Gerlach JH, Mirski SEL. Cancer Res 1991; 51: 3345. Cole SPC, Pinkoski MJ, Bhardwaj G, Deeley RG. Br J Cancer 1992; 65: 498. Cole SPC, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Stewart AJ, Kurz EU, Duncan AMV. Deeley RG. Science 1992: 258; 1650.
Amhm acid amidation motifs: a signal of biological activity
Cuttitta F, Quinn K, Englee-Miller MJ, Siegfried J, Unsworth E, Scott F, Vos M, Treston A, Mulshine J. National Cancer Institute, Biomarkers and Prevention Research Branch, 9610 Medical Center Drive, Suite 300, RockviNe, MD 20850.
Most peptide growth factors are generated from their respective larger precursor proteins via post-translational processing. Of the multiple enzymatic events that can occur during posttranslational processing, only proteolytic cleavage and amidation consistently track with bioactivity. A variety of substrate specific proteases are responsible for excising the peptide from its prepro molecule. Amidation involves a chemical alteration to the carboxy residue of a peptide’s C-terminal amino acid consisting of an -NH2 substitution of the -OH group. This modification is accomplished by sequential enzymatic steps and is initiated by specific amino acid motifs which are encoded within the precursor protein (Quinn et al., Cancer Cell 1991; 3: 504-510). We have used these motifs to analyze the precursors of established growth factors and have identified several previously unknown peptide amides with potential biological activity. Insulin-like growth factor-I (IGF-I) is 70-amino acid peptide which has been previously demonstrated to be a mitogen for both normal and malignant pulmonary cells. There are two messages which code for this peptide, IGF-IA and IGF-IB, as a result of alternative splicing. We have identified several amidation sequences in the precursor protein of the IGF-IB message and have observed the total absence of such motifs in the IGF-IA protein product. A synthetic homology (Y-23-RNH3 of one of these predicted peptide amides (IBE,) has been shown to stimulate the clonal growth of normal bronchial epithelial cells and a variety of pulmonary tumor cell lines (Siegfried et al. Proc Nat1 Acad Sci USA 1992; 89: 8107-8111. This peptide was shown to mediate its mitogenic effects on target cells through high affinity receptors (K,, = 1 x IO-“M) which are expressed at 1-2 x IO3 sites/cell. We propose that the IBE, peptide amide may function as an autocrine growth factor for small cell lung cancer (SCLC) since several tumor lines selectively express the IGF-IB message, have the enzyme repertoire for amidation to take place, have high affinity receptors for Y-23-R-NH2 and produce immune reactive proteinslpeptides with IBE, epitope expression. The use of amidation motifs to analyzing growth factor precursor molecules offers a new approach for identifying novel peptide amides with biological activity. These putative peptide can serve as potential target for early detection and prevention of malignant disease. A novel protein associated with cancer multidrug resistance Doyle LA. University of Maryland Cancer Center, Baltimore, MD.
We have noted expression of a novel 95-kDa membrane protein (P-95) associated with multidrug resistance in lung cancer and other human neoplasms. Overexpression of this protein was originally noted by Chen and Fojo in a subline of MCF-7 breast cancer cells selected in vitro for resistance to doxorubicin in the presence of verapamil, to prevent the development of P-glycoprotein overexpression (J Biol Chem
350 lines in athymic nude mice. Whereas cell lines derived from tumors with a non-small cell lung carcinoma-like phenotype were significantly hypomethylated relative to their parental cell lines, a cell line derived from a tumor lung carcinoma-like phenotype retained of its parental cell line. The data indicate tion of normally unmethylated CpG
with a more small cell the methylation status that de novo methylaisland sequences and
phenotype-associated demethylation of nonisland sequences accompany immortalization and oncogene-induced transformation of bronchial epithelial cells. Furthermore, at each locus tested, these altered methylation patterns occur at different stages during this progression. Because the in vitro model indicated a particular sensitivity of bronchial epithelial cells to methylation at chromosome 17~13.3, we next examined the status of the pYNZ22 locus in primary tissue. As expected for a CpG island, the pYNZ22 region was found unmethylated in normal whole lung tissue. However, vitro studies showed the pYNZ22 locus to be partially ated in cultured NHBE. To determine whether the
to be our in methylpartial
methylation in NHBE cells was induced by cell culture or preexisted in the primary epithelium, DNA from isolated bronchial epithelium and the underlying stromal layer was examined. Whereas the epithelial cell DNA was partially methylated at this locus, DNA from the stromal layer was unmethylated, indicating that the partial methylation of pYNZ22 was specific to the epithelial cells. This partial methylation also appeared to be a specific feature of bronchial epithelium since DNA from neither primary colonic epithelium nor colonic stroma was methylated at this locus. Studies addressing the possibilities that partial methylation of the pYNZ22 locus in bronchial epithelium might predispose the region to further methylation during lung tumorigenesis or that methylation specifically related to bronchial epithelial underway.
of this locus is development are
~53 and prognosis Carbone DP”, Mitsudomi Tb, Rusch Vc, Nowak JAd, Mcintire D”, Chiba lb. Gazdar AF”, Minna JD”, The Lung Cancer Study Group. “Simmons Cancer Center, UT Southwestern, Dallas, TX, ‘NCI-NMOB, Bethesda, MD, “Memorial Sloan Kettering Cancer Center, NY, NY, and ‘Illinois Masonic Medical Center, Chicago, IL. Abnormalities in the gene structure, protein structure, expression level, and other properties of p.53 have been found to be common in a wide range of human tumors. Many investigators have established that small cell lung cancers have mutation rates that approach 100X, and ~53 mutations are found in about 50% of non-small cell lung cancers (NSCLC). Such common abnormalities indicate significance in the molecular pathogenesis of cancer, but what is the clinical significance of these findings? The results are mixed. Some groups have reported association of p.53 mutations with worsened survival only in early stage patients, others only in late stage patients. Some groups have reported association of immunostaining positivity with adverse outcome, and others find no such association. Many of these studies are hampered by small numbers of patients, short followup, incomplete staging, hap-
Cancer 10 (1994) 347-373
hazard patient accrual, different techniques of analysis, ferent treatment strategies at different institutions.
and dif-
In our own series, we investigated the association of ~53 abnormalities and survival in 85 patients with NSCLC resected with curative intent in 1987 and 1988 as part of the Lung Cancer Study Group protocol 871. These patients were all systematically staged and their slides reviewed by reference pathologists. Our previous studies showed that only a subset of ~53 mutations in lung cancers result in overexpression and protein overexpression has been described in the absence of mutation. Therefore, we determined both protein overexpression (by immunostaining) and gene mutations (by single strand conformation polymorphism and DNA sequencing) in these tumors. Clinical followup data was available for 74 cases (median followup of 1564 days). Of the showed ~53 overexpression and 54% quences; however the concordance rate was a strong negative survival correlation
studied patients 55% had mutant ~53 sewas only 65%. There with positive ~53 im-
munostaining (P = O.Ol), but not with the presence of gene mutations (P = 0.96) in this group of patients. Why might immunopositivity be more significant than genetic mutations? Overexpression of ~53 may independently contribute to adverse outcome due to the ability of ~53 to act as a dominant oncogene (positive or negative), or alternatively, overexpression may reflect a response to ongoing DNA damage in the tumor, which in turn may be a marker for a more aggressive behavior. Thus in our series we find, in a single group of patients with over 4 years of follow-up, that ~53 overexpression, but not ~53 gene mutation, is a negative prognostic factor for patients with resected NSCLC. In summary, the disparity of results reported by different groups may reflect the fact that if such associations exist, they are relatively weak, or show up only with long follow-up. How could such a common lesion not have clinical relevance? One explanation may be that ~53 is only one component in a pathway which is universally abnormal in lung cancer, and when ~53 is normal, lesions in other as yet unknown genes may block its function. All lung cancers would thus be functionally equivalent with respect to this pathway and their behavior would be independent of the presence or absence of ~53 mutations. Even the finding of strong negative prognostic factors in lung cancer, however, may not allow us to translate these tindings into significant future improvements in survival with the ineffective or marginally effective adjuvant therapies currently available. Altered
drug distribution
and elevated expression of a novel
transporter gene (MRP) in a multidrug resistant lung cancer cell line
Cole SPC, Mirski
SEL, Bhardwaj
G, Almquist
KC, Deeley,
RG. Cancer Research Laboratories. Queen’s University. Kingston, Ontario K7L 3N6, Canada. Despite the widespread occurrence of resistance to chemotherapeutic agents in both small cell and non-small cell lung cancers, the clinically relevant molecular mechanisms responsible for this resistance remain largely unknown The role of P-glycoprotein, the I ‘IO-kDa plasma membrane protein encod-
4th LTBW Abstracts/
Lung Cancer IO (1994) 347-373
ed by the MDRl gene that is usually associated with multidrug resistance, appears limited in this tumour type. Our laboratory investigations have focused on a model system consisting of three cell lines: the NCI-H69 (H69) SCLC cell line; a doxorubicin-selected multidrug resistant variant, H69AR, that does not overexpress P-glycoprotein [1,2]; and a drug-sensitive revertant cell line, H69PR [3]. Laser-assisted confocal fluorescence microscopy studies show that daunomycin is predominantly localized in the nucleus and nuclear membrane of H69 cells; in contrast, the drug is sequestered in vesicle-like extranuclear structures in H69AR cells. These observations suggest that altered subcellular drug distribution may play a role in the resistance of the H69AR cell line. By screening an H69AR cDNA library by differential hybridization, we have recently identified a 65kb mRNA that is overexpressed approximately IOO-fold in H69AR cells compared to H69 and H69PR cells [4]. This mRNA is also expressed in five of five NSCLC cell lines tested. Southern blotting and cytogenetic studies indicate that the major mechanism underlying the elevated expression of this mRNA in H69AR cells is gene amplification. The 6%kb mRNA encodes a 1531 amino acid protein we have designated ‘multidrug resistance-associated protein’ (MRP). MRP is a novel member of the ATP-binding cassette (ABC) superfamily of transport systems. Other human proteins belonging to this family include the MHC class II-linked peptide transporters (TAP1 and TAP2), the cystic fibrosis transmembrane conductance regulator (CFTR) and the P-glycoproteins (MDRI and MDR3). However, the protein that is most closely related to MRP is ItpgpA, a gene which confers resistance to arsenite and trivalent antimonials in Leishmania farentolae. Increased expression of a 180- to I90-kDa 100000 x g membraneassociated protein is detected in H69AR cells using antibodies raised against several synthetic MRP peptides. The apparent molecular mass of this protein is about 20 kDa greater than Pglycoprotein and it is N-glycosylated. Photoaffinity labeling experiments with [32P]azido-ATP shows that it binds ATP. The isolation and characterization of MRP along with its increased expression in association with altered subcellular drug distribution provides the tirst definitive molecular evidence for the existence of a transporter that may confer or at least contribute to multidrug resistance in lung cancer cell lines. However, insight into the mechanism of the involvement of MRP in drug resistance awaits knowledge of the subcellular localization of the protein and the nature of the molecules it transports. Furthermore, a role for MRP in clinical drug resistance remains to be established. (Supported by MRC, NCIC and Cancer Research Sot.; SPCC is a Career Scientist of the OCTRF and RGD is the Queens University Stauffer Research Professor of Cancer Research.)
1 2 3 4
Mirski SEL, Gerlach JH, Cole SPC. Cancer Res 1987; 47: 2594. Cole SPC, Chanda ER, Dicke FP, Gerlach JH, Mirski SEL. Cancer Res 1991; 51: 3345. Cole SPC, Pinkoski MJ, Bhardwaj G, Deeley RG. Br J Cancer 1992; 65: 498. Cole SPC, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Stewart AJ, Kurz EU, Duncan AMV. Deeley RG. Science 1992: 258; 1650.
Amhm acid amidation motifs: a signal of biological activity
Cuttitta F, Quinn K, Englee-Miller MJ, Siegfried J, Unsworth E, Scott F, Vos M, Treston A, Mulshine J. National Cancer Institute, Biomarkers and Prevention Research Branch, 9610 Medical Center Drive, Suite 300, RockviNe, MD 20850.
Most peptide growth factors are generated from their respective larger precursor proteins via post-translational processing. Of the multiple enzymatic events that can occur during posttranslational processing, only proteolytic cleavage and amidation consistently track with bioactivity. A variety of substrate specific proteases are responsible for excising the peptide from its prepro molecule. Amidation involves a chemical alteration to the carboxy residue of a peptide’s C-terminal amino acid consisting of an -NH2 substitution of the -OH group. This modification is accomplished by sequential enzymatic steps and is initiated by specific amino acid motifs which are encoded within the precursor protein (Quinn et al., Cancer Cell 1991; 3: 504-510). We have used these motifs to analyze the precursors of established growth factors and have identified several previously unknown peptide amides with potential biological activity. Insulin-like growth factor-I (IGF-I) is 70-amino acid peptide which has been previously demonstrated to be a mitogen for both normal and malignant pulmonary cells. There are two messages which code for this peptide, IGF-IA and IGF-IB, as a result of alternative splicing. We have identified several amidation sequences in the precursor protein of the IGF-IB message and have observed the total absence of such motifs in the IGF-IA protein product. A synthetic homology (Y-23-RNH3 of one of these predicted peptide amides (IBE,) has been shown to stimulate the clonal growth of normal bronchial epithelial cells and a variety of pulmonary tumor cell lines (Siegfried et al. Proc Nat1 Acad Sci USA 1992; 89: 8107-8111. This peptide was shown to mediate its mitogenic effects on target cells through high affinity receptors (K,, = 1 x IO-“M) which are expressed at 1-2 x IO3 sites/cell. We propose that the IBE, peptide amide may function as an autocrine growth factor for small cell lung cancer (SCLC) since several tumor lines selectively express the IGF-IB message, have the enzyme repertoire for amidation to take place, have high affinity receptors for Y-23-R-NH2 and produce immune reactive proteinslpeptides with IBE, epitope expression. The use of amidation motifs to analyzing growth factor precursor molecules offers a new approach for identifying novel peptide amides with biological activity. These putative peptide can serve as potential target for early detection and prevention of malignant disease. A novel protein associated with cancer multidrug resistance Doyle LA. University of Maryland Cancer Center, Baltimore, MD.
We have noted expression of a novel 95-kDa membrane protein (P-95) associated with multidrug resistance in lung cancer and other human neoplasms. Overexpression of this protein was originally noted by Chen and Fojo in a subline of MCF-7 breast cancer cells selected in vitro for resistance to doxorubicin in the presence of verapamil, to prevent the development of P-glycoprotein overexpression (J Biol Chem