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We have identified a novel pathway of ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) signaling that results in nuclear factor B (NF-B) activation and chemoresistance in response to DNA damage. We show that the anthracycline doxorubicin (DOX) and its congener N-benzyladriamycin (AD 288) selectively activate ATM and DNA-PK, respectively. Both ATM and DNA-PK promote sequential activation of the mitogen-activated protein kinase (MAPK)/p90 rsk signaling cascade in a p53-independent fashion. In turn, p90 rsk interacts with the IB kinase 2 (IKK-2) catalytic subunit of IKK, thereby inducing NF-B activity and cell survival. Collectively, our findings suggest that distinct members of the phosphatidylinositol kinase family activate a common prosurvival MAPK/IKK/NF-B pathway that opposes the apoptotic response following DNA damage.

The majority of the protein mass of HeLa 40S heterogeneous nuclear ribonucleoprotein monoparticles is composed of multiple copies of six proteins that resolve in SDS gels as three groups of doublet bands (A1, A2; B1, B2; and C1, C2) (13eyer, A. L., M. E. Christensen, 13. W. Walker, and W. M. LeStourgeon. 1977. Cell. 11: 127-138). We report here that when 40S monoparticles are exposed briefly to ribonuclease, proteins A1, C1, and C2 are solubilized coincidentally with the loss of most premessenger RNA sequences. The remaining proteins exist as tetramers of (A2)3(B1) or pentamers of (A2)3(B1)(132). The tetramers may reassociate in highly specific ways to form either of two different structures. In 0.1 M salt approximately 12 tetramers (derived from three or four monoparticles) reassemble to form highly regular structures, which may possess dodecahedral symmetry. These structures sediment at 43S, are 20-22 nm in width, and have a mass near 2.3 million. These structures possess 450-500 bases of slowly labeled RNA, which migrates in gels as fragments 200-220 bases in length. In 9 mM salt the tetramers reassociate to form 2.0 M salt-insoluble helical filaments of indeterminant length with a pitch near 60 nm and diameter near 18 nm. If 40S monoparticles are treated briefly with nuclease-free proteases, the same proteins solubilized by nuclease (A1, C1, and C2) are preferentially cleaved. This protein cleavage is associated with the dissociation of most of the heterogeneous nuclear RNA. Proteins A2 and B1 again reassemble to form uniform, globular particles, but these sediment slightly slower than intact monoparticles. These findings indicate that proteins A1, C1, and C2 and most of the premessenger sequences occupy a peripheral position in intact monoparticles and that their homotypic and heterotypic associations are dependent on protein-RNA interactions. Protein cross-linking studies demonstrate that trimers of A1, A2, and C1 exist as the most easily stabilized homotypic association in 40S particles. This supports the 3:1 ratio (via densitometry) of the A and C proteins to the B proteins and indicates that 40S monoparticles are composed of three or four repeating units, each containing 3(A1),3(A2),1(B1),1(B2),3(C1), and 1 (C2).When isolated nuclei from mammalian cells are disrupted briefly with ultrasound (1-3) or extracted with low ionic strength buffers (4, 5), the majority of the premessenger RNA sequences (heterogeneous nuclear [hn] RNA) ~ are recovered Abbreviations used in this paper. CuP, (orthophenanthrolinehCu(II) complex; DTBP, 3,3'-dimethyl dithiobispropionimidate dihydrochloride; hnRNA, heterogeneous nuclear RNA; hnRNP, heteroge-1570 in 20-25-nm 40S ribonucleoprotein complexes or monoparticles (1-13). Along with hnRNA, these monoparticles are primarily composed of multiple copies of six nucleus-specific polypeptides that migrate in SDS polyacrylamide gels as three groups of doublet bands (A1,A2; B1,B2, and C1,C2) with neous nuclear ribonucleoprotein; NRS, nuclease-resistant structure; OD, optical ...

In multidrug-resistant mouse J774.2 cells, the differential overproduction of functionally distinct phosphoglycoprotein isoforms reflects the amplification or transcriptional activation or both of two mdr gene family members, mdrla and mdrlb. The mdrla gene is a complex transcriptional unit whose expression is associated with multiple transcript sizes. Independently selected multidrug-resistant J774.2 cell lines differentially overexpress either 4.6-and 5.0-kilobase (kb) or 4.7-and 5.1-kb mdrla transcripts. However, abundant overproduction of the mdrla gene product was observed only in cell lines which overexpressed the 4.6-and 5.0-kb mRNAs. In order to determine the basis for mdrla transcript heterogeneity and the relationship between transcript size and steady-state mdrla protein levels, genomic and cDNA sequence analyses of the 5' and 3' ends of the mdrla gene were carried out. Promoter sequence analysis and primer extension mapping indicated that mdrla transcripts were differentially initiated from two putative promoters to generate either 5.1-and 4.7-kb or 5.0-and 4.6-kb transcripts in four multidrug-resistant J774.2 cell lines. Sequence analysis of 3' cDNA variants and a 3' genomic fragment revealed that the 5.1-and 5.0-kb mRNAs had identical 3'-untranslated regions which differed from those of the 4.7-and 4.6-kb mRNAs as a result of the utilization of a more downstream alternative poly(A) addition signal. Transcript initiation from the putative upstream promoter correlated with a 70 to 85% decrease in steady-state mdrla protein levels relative to transcript levels. In addition, the identification of putative AP-1 and AP-2 promoter elements suggests a possible role for protein kinase A and protein kinase C in the regulation of mdrla. The implications of these findings for mdr gene expression and regulation are discussed.Acquired or inherent resistance of human tumors to a wide variety of structurally and mechanistically unrelated antineoplastic agents frequently results in treatment failure (13). Rodent and human cell lines selected in vitro for resistance to a single cytotoxic drug have been developed as model systems for studying the molecular basis of multidrug resistance (11,52). These cell lines exhibit cross-resistance to a broad spectrum of structurally and functionally diverse lipophilic cytotoxic compounds derived primarily from natural products, such as plant alkaloids and antibiotics of fungal origin. The basis for the multidrug resistance (MDR) phenotype appears to involve decreased net intracellular accumulation of drugs due to an energy-dependent drug efflux (9,25,45) which correlates with the increased expression of mdr genes. In humans and rodents, mdr genes comprise a small gene family of two (mdrl and mdr2) and three (mdrla, mdrlb, and mdr2) members, respectively (6,23,35). Increased expression of the mdrl-class genes, a single mdrl gene in humans (44,51), and the mdrla and/or mdrlb gene in rodents (23), often accompanied by gene amplification, results in the overproduction of a family of...