Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA1
Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany2
Author for correspondence: Stephen Alexander. Tel: +1 573 882 6670. Fax: +1 573 882 0123. e-mail: alexanderst{at}missouri.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: ceramide, sphingosine 1-phosphate, chemotherapy, drug-resistance, PKA
Abbreviations: MMS, methyl methanesulfonate; PIP5K, phosphatidylinositol-4-phosphate 5-kinase; PKA, protein kinase A; REMI, restriction-enzyme-mediated integration; S-1-P, sphingosine 1-phosphate
The GenBank accession numbers for the sequences reported in this paper are AF233610 (S-1-P lyase), AF233612 (PIP5K), AF233611 (P2Y purine receptor 1), AF233613 (CAAX prenyl protease) and AF233614 (unidentified gene).
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An important example is illustrated in the case of cisplatin [cis-diamminedichloroplatinum(II)] and its derivatives, which are widely used anti-cancer drugs (Chu, 1994 ; Eastman, 1986
; Lippard, 1982
). Cisplatin is used alone or in combination with other drugs for many types of human malignancies, including testicular, ovarian, bladder, cervical, head and neck, oesophageal and some lung cancers (Loehrer & Einhorn, 1984
). There is general agreement that the effect of cisplatin is due to its ability to form intrastrand cross-links between adjacent purines in DNA (Sherman & Lippard, 1987
; Takahara et al., 1995
). However, its therapeutic efficacy is frequently limited by the development of drug-resistant tumour cell populations. In addition, cisplatin has minimal effect against common tumours such as colorectal and pancreatic (Perez, 1998
).
Numerous mechanisms have been suggested for the development of cellular resistance to cisplatin. Some include changes in the cellular drug concentration resulting from decreased accumulation, increased efflux or increased inactivation of the drug (Andrews & Howell, 1990 ; Chu, 1994
). Other studies have focused on proteins that affect the recognition of cisplatin damage, such as the high mobility group proteins (Kohn, 1999
; Ohndorf et al., 1999
) or proteins that affect mismatch repair such as the Mut family of proteins (Drummond et al., 1996
; Fink et al., 1997
, 1998
). Specific molecules have been implicated in cisplatin-induced cell death or resistance, such as p53 and p73, and the tyrosine kinase c-Abl, which has been related to the p73 response to cisplatin (Agami et al., 1999
; Gong et al., 1999
; Yuan et al., 1999
). All of these studies were focused on genes and mechanisms which were a priori suspected to modulate the cellular response to the drug.
In this report we used a direct genetic approach, employing insertional mutagenesis, to specifically identify novel genetic pathways that are involved in the cellular response and resistance to cisplatin in Dictyostelium discoideum. This relatively simple multicellular organism has strong evolutionary conservation with higher eukaryotes (Baldauf & Doolittle, 1997 ; Kessin, 1997
). It is widely used in studies on cell and developmental biology because of its multicellularity (Alexander & Rossomando, 1992
; Loomis, 1982
) and the availability of powerful molecular genetic approaches (Kuspa & Loomis, 1992
; Spudich, 1987
).
Interestingly, the genes that we identified as responsible for cisplatin resistance are not directly involved in drug import or efflux, in DNA repair or in multi-drug resistance. Some define signal transduction pathways that regulate cell death, cell proliferation or gene regulation in other systems. Importantly, some of the mutants exhibit abnormal developmental phenotypes, demonstrating that the genes that are involved in responding to DNA damage also function in normal development. The power of this genetic approach is that it identifies individual genes and pathways previously unsuspected of having a direct link to cisplatin resistance and offers possible ways to manipulate the cellular response to the drug.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Restriction-enzyme-mediated integration (REMI) mutagenesis.
Cells were mutagenized by electroporation with the pUCBsrBam plasmid (a gift from K. Saxe, Emory University School of Medicine) (Bear et al., 1998
), which contains the gene for blasticidin S resistance (bsr) (Sutoh, 1993
), using established methods (Kuspa & Loomis, 1992
). Based on our experimental determination of transformation efficiency, we estimate that we examined about 50006000 individual insertional mutants in this experiment. Based on current estimates of the total number of genes in Dictyostelium, and the frequency of obtaining developmental mutants (Shaulsky et al., 1996
), this represents coverage of about 1015% of the genome.
Transformed cells were selected for growth in HL-5 medium containing 10 µg blasticidin ml-1 (ICN). The blasticidin-resistant transformants were then selected for growth in 300 µM cisplatin, the maximum concentration that can be added to the cells (Sigma). Surviving cells were plated for single colonies on SM agar plates (Sussman, 1987 ) and clones were picked and re-tested for their ability to grow in the presence of 300 µM cisplatin. The disruption vector and the flanking fragments of the disrupted genes were then excised, ligated, cloned into bacteria and subsequently used for homologous recombination to recapitulate the mutation.
Sequence identification.
Both genomic fragments flanking the disruption vector were sequenced, using vector-specific primers. The sequences were then checked against sequences in the GenBank database using the BLAST program, as well as against the sequence data generated by the Dictyostelium cDNA and genomic sequencing project (Morio et al., 1998 ; http://dicty.cmb.nwu.edu/Dicty/dictyostelium_genomics.htm). In most cases we were able to assemble the majority of the gene.
Cell survival assays.
Cell survival after exposure to damaging agents was determined by plating aliquots of serially diluted cells along with Klebsiella aerogenes on SM plates (Sussman, 1987 ). Each surviving cell gives rise to a single plaque on the bacterial lawn. Survival was tested after treatments with cisplatin, the oxidizing agent H2O2 and the alkylating agent methyl methanesulfonate (MMS) (all from Sigma), as well as after treatment with UV light. For cisplatin, stock solutions were made by dissolving cisplatin in Pt buffer (3 mM NaCl, 1 mM sodium phosphate, pH 7·4), to a final concentration of 3·3 mM and verified by checking the absorbance at 220 nm (based on an extinction coefficient at 220 nm of 1·957 mM-1 cm-1). H2O2 was used for 1 h at the indicated concentrations, based on an extinction coefficient at 240 nm of 43·6 M-1 cm-1 (Garcia et al., 2000
). MMS was directly diluted into medium from an 11·9 M stock solution, supplied by the distributor, and cells were incubated for 1 h. UV exposure was performed in LPS/5 mM EDTA at indicated fluences, adjusted according to Morowitz (Morowitz, 1950
; Yu et al., 1998
).
Molecular biology techniques.
Standard techniques for molecular cloning, Southern and Northern analyses and PCR were used (Lee et al., 1997 ; Sambrook et al., 1989
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The excised fragments were sequenced and we were able to identify the affected genes in six of the seven mutant strains (Table 1.)
|
Gene 2. regA encodes a bipartite cAMP phosphodiesterase in Dictyostelium (Shaulsky et al., 1996 ), which is composed of a regulatory response domain and a phosphodiesterase domain that is 49% similar to the Drosophila dunce gene (Davis & Kiger, 1981
; Walter & Kiger, 1984
).
Gene 3.
Golvesin is a novel, previously identified Dictyostelium gene. Its gene product associates with the Golgi apparatus and intracellular vesicles (GenBank accession no. AAC16756).
Gene 4.
This gene is 45% similar to the kinase domain within a putative phosphatidylinositol-4-phosphate 5-kinase (PIP5K) from Arabidopsis (accession no. AAC78530) and may represent a novel member of this family of kinases, which are involved in the synthesis of phosphatidylinositol 4,5-bisphosphate (Loijens et al., 1996 ).
Gene 5.
The gene has 52% similarity to the human P2Y purine receptor 1 (Leon et al., 1996 ) within the first third of the gene for which we have sequence.
Gene 6. The disruption in this strain occurred 280 bp upstream of the gene which is 51% similar to the human CAAX prenyl protease gene (Tam et al., 1998 ).
Gene 7.
The insertion in this strain occurred in an AT-rich intergenic region, which we have been unable to assign to a specific gene.
Cisplatin resistance
The resistance of these mutant strains to cisplatin was quantified for both the original mutant clones and the homologous recombinants. We determined viability after cells were exposed to a single dose of 300 µM cisplatin for increasing periods of time up to 24 h and the results are presented in Fig. 1(a) and Table 1
. Increasing the time of exposure to cisplatin does not result in further cell death or the selection of heritable mutants (Yu et al., 1998
). The resistance of the strains produced by homologous recombination was virtually identical to the resistance of the original mutant clones obtained directly from the REMI mutagenesis (data not shown). In addition, we tested independent disruption mutants for the golvesin and regA genes (GenBank accession no. AAC16756; Shaulsky et al., 1996
). Although the disruptions in these strains were in different domains from the disruptions we identified, these mutant strains had identical levels of increased resistance when compared to their wild-type parents (data not shown). All the mutants showed increasing resistance to cisplatin, ranging from 4- to 25-fold. These levels of resistance are equal to or higher than the levels observed in cisplatin-resistant animal cell lines (Katabami et al., 1992
; Kuppen et al., 1988
).
|
The resistance of these cells to cisplatin is not due to a fundamental change in the growth rate of the cells. The growth rate of each of these mutant strains in normal medium was identical to that of the wild-type parent (Fig. 1c).
Developmental abnormalities
Interestingly, three of the mutants had immediately obvious defects in development. The normal size and proportioning of wild-type fruiting bodies are shown in Fig. 2(a), where a mass of spores rests on top of the cellular stalk. The S-1-P lyase null mutant produced short fruiting bodies with a dramatically thicker stalk and a drastically reduced number of spores (Fig. 2b
). The regA null mutant produced fruiting bodies with spore masses that cannot rise up the stalk during morphogenesis (Fig. 2c
). It is identical in phenotype to the regA mutant that was produced by Shaulsky et al. (1998)
. The PIP5K null mutant produced fruiting bodies with apparently altered cell-type proportions. It has abnormally long stalks and a spore mass of reduced size (Fig. 2d
).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have taken a direct genetic approach to identifying biochemical pathways that are involved directly in cisplatin resistance by selecting mutants that are resistant to the drug. This approach has the advantage of being unbiased and indeed has resulted in the identification of six genes previously unsuspected of having a role in the cellular response to this drug. Since the mutant screen was not saturating, we expect that additional cisplatin resistance genes will be identified in the future. Indeed, a multi-drug-resistant transporter has been recently identified in Dictyostelium (Good & Kuspa, 2000 ) and it will be interesting to test whether it functions in modulating cisplatin sensitivity. Two of the genes we have identified in this study immediately suggest testable mechanisms that can explain the increased sensitivity to cisplatin.
In the case of S-1-P lyase, complex sphingolipids are known to play a role in cell proliferation and survival (Merrill et al., 1993 ). Recently, the products of sphingomyelin catabolism (ceramide, sphingosine and S-1-P) have been implicated in signal transduction processes in animal cells, presumably acting as second intracellular messengers. S-1-P lyase catalyses the conversion of S-1-P to hexadecanal and phosphoethanolamine. It has been suggested that the pathway that begins with the degradation of sphingomyelin to ceramide is involved in cell death and that the modulation of ceramide and S-1-P levels acts as a rheostat, maintaining a balance between proliferation and cell death functions (Spiegel, 1999
). The work described herein is the first to demonstrate a relationship between cisplatin resistance and the cellular levels of S-1-P. It will be important to demonstrate that the other members of this pathway can influence resistance to cisplatin. In this regard we have already identified the sphingosine kinase gene and are working to make a disruption in this gene. We expect it will have increased sensitivity to cisplatin due to the decrease in S-1-P level. Overall, this suggests that manipulating the levels of S-1-P in the cells could be an important therapeutic avenue by potentiating tumour cells to be more sensitive to cisplatin or other drugs.
In the case of regA, the gene product was recently identified as a central component in the pathway for spore differentiation in Dictyostelium (Shaulsky et al., 1996 ). It has a Drosophila homologue, the dunce gene, which has been related to learning and memory in this organism (Byers et al., 1981
; Davis & Kiger, 1981
). The RegA protein is a cAMP phosphodiesterase, regulating the cAMP level in prespore cells, which in turn regulates protein kinase A (PKA). The Dictyostelium PKA exists as a dimer of one regulatory and one catalytic subunit, while the mammalian enzyme consists of two subunits of each. The binding of cAMP to the regulatory subunit (PKA-R) releases and activates the catalytic subunit (PKA-C). The developmental analysis of regA has been extensive and it is known that the expression of the RegA-, PKA-C- and PKA-R-encoding genes increases during development. There is a low level of expression of all three genes in growing cells, but none is required for growth (Loomis, 1998
). The discovery of this gene in our selection was important because it suggests that cisplatin resistance may be linked to PKA signalling pathways. The role of PKA in response to cisplatin can be easily tested because of the wide availability of mutants altered in PKA activity in Dictyostelium (Loomis, 1998
). In recent work PKA has been linked to cisplatin resistance in CHO cells (Cvijic et al., 1998a
), but has been ruled out as having a role in response to cisplatin in yeast (Cvijic et al., 1998b
). Additionally, an elevated level of cAMP has been shown to cause attenuation of the response to cisplatin in macrophages (von Knethen et al., 1998
).
There is an increasing number of examples of molecular cross-talk in DNA repair, including the sharing of enzymic components between nucleotide excision repair, base excision repair and mismatch repair mechanisms (Sancar, 1999 ; Swanson et al., 1999
). In Dictyostelium, at least eight non-allelic mutants that were isolated based on increased sensitivity to
-irradiation also showed increased sensitivities to other DNA-damaging drugs (Bronner et al., 1992
; Podgorski & Deering, 1980
). The results of our mutant selection clearly illustrate that multiple genes and pathways are involved in the response to cisplatin. However, the deletion of S-1-P lyase-, RegA- and PIP5K-encoding genes did not result in resistance to other DNA-damaging agents in addition to cisplatin. The specificity for resistance to cisplatin in Dictyostelium is similar to that observed with several cisplatin-resistant tumour cell lines (Teicher et al., 1991
; Rabo et al., 1996
) and is important because it offers specific potential target pathways for chemotherapeutic agents.
The identification of the developmental phenotypes associated with some of the cisplatin-resistant mutants was an important outcome of this study. Although the role of regA in development has been well studied, nothing is known about the other two genes. The severity of the S-1-P lyase mutant is particularly interesting and we have already shown that it produces very few mature spores. Further work is needed to fit the action of these genes into the increasingly well understood gene circuitry that is already known to control development in Dictyostelium (Laurence & Firtel, 1999 ).
Dictyostelium is more often associated with studies of cellular and developmental biology, such as the control of cell chemotaxis and motility (Parent & Devreotes, 1999 ) and regulated protein secretion (Srinivasan et al., 2000
). However, it is clear from the work described here that it can be effectively used to discover genes underlying cellular responses to important pharmacological agents. Moreover, its unique biology allows the immediate identification of those genes that also have a role in multicellular development as well as in the response to the drug.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alexander, S. & Rossomando, E. F. (1992). Regulation of morphogenesis in Dictyostelium discoideum. In Morphogenesis: An Analysis of the Development of Biological Form , pp. 29-61. Edited by E. F. Rossomando & S. Alexander. New York: Marcel Dekker.
Andrews, P. A. & Howell, S. B. (1990). Cellular pharmacology of cisplatin: perspectives on mechanisms of acquired resistance. Cancer Cells 2, 35-43.[Medline]
Baldauf, S. L. & Doolittle, W. F. (1997). Origin and evolution of the slime molds (Mycetozoa). Proc Natl Acad Sci USA 94, 12007-12012.
Bear, J. E., Rawls, J. F. & Saxe, C. L.3rd (1998). SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development. J Cell Biol 142, 1325-1335.
Bronner, C. E., Welker, D. L. & Deering, R. A. (1992). Mutations affecting sensitivity of the cellular slime mold Dictyostelium discoideum to DNA-damaging agents. Mutat Res DNA Repair 274, 187-200.[Medline]
Byers, D., Davis, R. L. & Kiger, J. A.Jr (1981). Defect in cyclic AMP phosphodiesterase due to the dunce mutation of learning in Drosophila melanogaster. Nature 289, 79-81.[Medline]
Chu, G. (1994). Cellular responses to cisplatin. J Biol Chem 269, 787-790.
Cocucci, S. & Sussman, M. (1970). RNA in cytoplasmic and nuclear fractions of cellular slime mold amoebas. J Cell Biol 45, 399-407.
Cvijic, M. E., Yang, W. L. & Chin, K. V. (1998a). Cisplatin resistance in cyclic AMP-dependent protein kinase mutants. Pharmacol Ther 78, 115-128.[Medline]
Cvijic, M. E., Yang, W. L. & Chin, K. V. (1998b). Cisplatin sensitivity in cAMP-dependent protein kinase mutants of Saccharomyces cerevisiae. Anticancer Res 18, 3187-3192.[Medline]
Davis, R. L. & Kiger, J. A.Jr (1981). Dunce mutants of Drosophila melanogaster: mutants defective in the cyclic AMP phosphodiesterase enzyme system. J Cell Biol 90, 101-107.[Abstract]
Drummond, J. T., Anthoney, A., Brown, R. & Modrich, P. (1996). Cisplatin and adriamycin resistance are associated with MutL alpha and mismatch repair deficiency in an ovarian tumour cell line. J Biol Chem 271, 19645-19648.
Eastman, A. (1986). Re-evaluation of interaction of cis-dichloro(ethylenediamine)platinum(II) with DNA. Biochemistry 25, 3912-3915.[Medline]
Fink, D., Nebel, S., Norris, P. S., Baergen, R. N., Wilczynski, S. P., Costa, M. J., Haas, M., Cannistra, S. A. & Howell, S. B. (1998). Enrichment for DNA mismatch repair-deficient cells during treatment with cisplatin. Int J Cancer 77, 741-746.[Medline]
Fink, D., Zheng, H., Nebel, S. & 8 other authors (1997). In vitro and in vivo resistance to cisplatin in cells that have lost DNA mismatch repair. Cancer Res 57, 18411845.[Abstract]
Friedberg, E. C., Walker, G. C. & Siede, W. (1995). DNA Repair and Mutagenesis. Washington, DC: American Society for Microbiology.
Garcia, M. X. U., Foote, C., van Es, S., Devreotes, P. N., Alexander, S. & Alexander, H. (2000). Differential developmental expression and cell type specificity of Dictyostelium catalases and their response to oxidative stress and UV-light. Biochim Biophys Acta 1492, 295310.[Medline]
Gong, J., Costanzo, A., Yang, H., Melino, G., Kaelin, W. G., Levrero, M. & Wang, J. Y. J. (1999). The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature 399, 806-809.[Medline]
Good, J. R. & Kuspa, A. (2000). Evidence that a cell-type-specific efflux pump regulates cell differentiation in Dictyostelium. Dev Biol 220, 53-61.[Medline]
Katabami, M., Fijita, H., Haneda, H., Akita, H., Kuzumaki, N., Miyamoto, H. & Kawakami, Y. (1992). Reduced drug accumulation in a newly established human lung squamous-carcinoma cell line resistant to cis-diamminedichloroplatinum(II). Biochem Pharmacol 44, 394-397.[Medline]
Kessin, R. H. (1997). The evolution of the cellular slime mold. In Dictyostelium A Model System for Cell and Developmental Biology , pp. 349-362. Edited by Y. Maeda, K. Inouye & I. Takeuchi. Tokyo: Universal Academy Press.
von Knethen, A., Lotero, A. & Brune, B. (1998). Etoposide and cisplatin induced apoptosis in activated RAW 264.7 macrophages is attenuated by cAMP-induced gene expression. Oncogene 17, 387-394.[Medline]
Kohn, K. W. (1999). Molecular interaction map of the mammalian cell cycle control and DNA repair systems. Mol Biol Cell 10, 2703-2734.
Kuppen, P. J., Schuitemaker, H., vant Veer, L. J., de Bruijn, E. A., van Oosterom, A. T. & Schrier, P. I. (1988). cis-Diamminedichloroplatinum(II)-resistant sublines derived from two human ovarian tumour cell lines. Cancer Res 48, 3355-3359.[Abstract]
Kuspa, A. & Loomis, W. F. (1992). Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. Proc Natl Acad Sci USA 89, 8803-8807.[Abstract]
Laurence, A. & Firtel, R. (1999). Integration of signalling networks that regulate Dictyostelium differentiation. Annu Rev Cell Dev Biol 15, 469-517.[Medline]
Lee, S.-K., Yu, S.-L., Garcia, M. X., Alexander, H. & Alexander, S. (1997). Differential developmental expression of the repB and repD xeroderma pigmentosum related DNA helicase genes from Dictyostelium discoideum. Nucleic Acids Res 25, 2365-2374.
Leon, C., Vial, C., Cazenave, J. P. & Gachet, C. (1996). Cloning and sequencing of a human cDNA encoding endothelial P2Y1 purinoceptor. Gene 171, 295-297.[Medline]
Lippard, S. J. (1982). New chemistry of an old molecule: cis-[Pt(NH3)2Cl2]. Science 218, 1075-1082.[Medline]
Loehrer, P. J. & Einhorn, L. H. (1984). Cisplatin. Ann Intern Med 100, 704-713.[Medline]
Loijens, J. C., Boronenkov, I. V., Parker, G. J. & Anderson, R. A. (1996). The phosphatidylinositol 4-phosphate 5-kinase family. Adv Enzyme Regul 36, 115-140.[Medline]
Loomis, W. F. (1982). The Development of Dictyostelium discoideum. New York: Academic Press.
Loomis, W. F. (1998). Role of PKA in the timing of developmental events in Dictyostelium cells. Microbiol Mol Biol Rev 62, 684-694.
Los, G., Johnson, A., Yang, F., Berry, C. & Howell, S. (1998). Identification of genes differentially expressed in cisplatin-sensitive versus resistant tumour cells. In The Microarray Meeting: Technology, Application and Analysis, p. 60. Scottsdale, AZ: Nature America.
Merrill, A. H. J., Hunnun, Y. A. & Bell, R. M. (1993). Introduction: sphingolipids and their metabolites in cell regulation. Adv Lipid Res 25, 1-24.[Medline]
Morio, T., Urushihara, H., Saito, T. & 14 other authors (1998). The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development. DNA Res 5, 335340.[Medline]
Morowitz, H. J. (1950). Absorption effects in volume irradiation of microorganisms. Science 111, 229-230.
Ohndorf, U. M., Rould, M. A., He, Q., Pabo, C. O. & Lippard, S. J. (1999). Basis for recognition of cisplatin-modified DNA by high-mobility-group proteins. Nature 399, 708-712.[Medline]
Parent, C. A. & Devreotes, P. N. (1999). A cells sense of direction. Science 284, 765-770.
Perez, R. P. (1998). Cellular and molecular determinants of cisplatin resistance. Eur J Cancer 34, 1535-1542.[Medline]
Podgorski, G. & Deering, R. A. (1980). Effect of methyl methanesulfonate on survival of radiation-sensitive strains of Dictyostelium discoideum. Mutat Res 73, 415-418.[Medline]
Rabo, Y. B., Shoshan, M. C., Linder, S. & Hansson, J. (1996). Different mechanisms are responsible for c-jun mRNA induction by cisplatin and ultraviolet light. Int J Cancer 65, 821-826.[Medline]
Saba, J. D., Nara, F., Bielawska, A., Garrett, S. & Hannun, Y. A. (1997). The BST1 gene of Saccharomyces cerevisiae is the sphingosine-1-phosphate lyase. J Biol Chem 272, 26087-26090.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sancar, A. (1999). Excision repair invades the territory of mismatch repair. Nat Genet 21, 247-249.[Medline]
Scagliotti, G. V., Novello, S. & Selvaggi, G. (1999). Multidrug resistance in non-small-cell lung cancer. Ann Oncol 10, S83-S86.[Medline]
Shaulsky, G., Escalante, R. & Loomis, W. F. (1996). Developmental signal transduction pathways uncovered by genetic suppressors. Proc Natl Acad Sci USA 93, 15260-15265.
Shaulsky, G., Fuller, D. & Loomis, W. F. (1998). A cAMP-phosphodiesterase controls PKA-dependent differentiation. Development 125, 691-699.
Sherman, S. E. & Lippard, S. J. (1987). Structural aspects of platinum anticancer drug interactions with DNA. Chem Rev 87, 1153-1181.
Soll, D. (1987). Methods for manipulating and investigating developmental timing in Dictyostelium discoideum. Methods Cell Biol 28, 413-431.[Medline]
Spiegel, S. (1999). Sphingosine 1-phosphate: a prototype of a new class of second messengers. J Leukoc Biol 65, 341-344.[Abstract]
Spudich, J. (1987). Dictyostelium discoideum: molecular approaches to cell biology. Methods Cell Biol 28, 1-516.[Medline]
Srinivasan, S., Alexander, H. & Alexander, S. (2000). Crossing the finish line of development: regulated secretion and assembly of the Dictyostelium spore coat proteins. Trends Cell Biol 10, 215-219.[Medline]
Sussman, M. (1987). Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. Methods Cell Biol 28, 9-29.[Medline]
Sutoh, K. (1993). A transformation vector for Dictyostelium discoideum with a new selectable marker bsr. Plasmid 30, 150-154.[Medline]
Swanson, R. L., Morey, N. J., Doetsch, P. W. & Jinks-Robertson, S. (1999). Overlapping specificities of base excision repair, nucleotide excision repair, recombination, and translesion synthesis pathways for DNA base damage in Saccharomyces cerevisiae. Mol Cell Biol 19, 2929-2935.
Takahara, P. M., Rosenzweig, A. C., Frederick, C. A. & Lippard, S. J. (1995). Crystal structure of double-stranded DNA containing the major adduct of the anticancer drug cisplatin. Nature 377, 649-652.[Medline]
Tam, A., Nouvet, F. J., Fujimura-Kamada, K., Slunt, H., Sisodia, S. S. & Michaelis, S. (1998). Dual role for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing. J Cell Biol 142, 635-649.
Teicher, B. A., Holden, S. A., Herman, T. S., Sotomayop, E., Khandekar, V., Rosbe, K. W., Brann, T. W., Korbut, T. T. & Frei, E.III (1991). Characteristics of five human tumour cell lines and sublines resistant to cis-diamminedichloroplatinum(II). Int J Cancer 47, 252-260.[Medline]
Walter, M. F. & Kiger, J. A.Jr (1984). The dunce gene of Drosophila: roles of Ca2+ and calmodulin in adenosine 3':5'-cyclic monophosphate-specific phosphodiesterase activity. J Neurosci 4, 495-501.[Abstract]
Yu, S. L., Lee, S. K., Alexander, H. & Alexander, S. (1998). Rapid changes of nucleotide excision repair gene expression following Uv-irradiation and cisplatin treatment of Dictyostelium discoideum. Nucleic Acids Res 26, 3397-3403.
Yuan, Z. M., Shioya, H., Ishiko, T. & 7 other authors (1999). p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage. Nature 399, 814817, erratum 400, 792.[Medline]
Received 13 April 2000;
revised 15 June 2000;
accepted 20 June 2000.