I. Introduction

Bleomycin is an antibiotic originally isolated four decades ago from Streptomyces verticillis (Umezawa, 1965; Umezawa et al, 1966). Subsequent studies clearly demonstrated that bleomycin can diminish the growth of experimentally induced tumors in mice and rats, and dramatically decrease the size of human tumors (Suzuki et al, 1968, 1970a; Ichikawa et al, 1969; Kanno et al, 1969; Oka et al, 1970; Terasima and Umezawa, 1970). Additional studies demonstrated that bleomycin possesses the ability to induce micronuclei formation and chromosome aberrations in human lymphocytes, as well as mitotic recombination and mutations in many organisms (Suzuki et al, 1970b; Terasima et al, 1970; Hoffmann et al, 1993). Collectively, these observations led to the suggestion that bleomycin mediates its chemotherapeutic effect by directly destroying the DNA and causing cell death. Following these findings, the detailed mechanism by which bleomycin induces DNA lesions began to unfold (Burger et al, 1981, 1982; Hecht, 1986; Kane and Hecht, 1994). Besides its action on DNA, bleomycin can also selectively destroy RNA raising the possibility that bleomycin-induced cell death might be a contribution from the destruction of more than one target (Carter et al, 1990; Huttenhofer et al, 1992; Holmes and Hecht, 1993, 1994; Morgan and Hecht, 1994; Keck and Hecht, 1995). However, accessing these targets is complicated by the fact that bleomycin is a large hydrophilic molecule that is first channel to the vacuoles, presumably for detoxification (Aouida et al, 2004a), and perhaps accumulated to a threshold level where it might cause oxidation of the vacuolar inner membrane and subsequent release into the cytoplasm (Ekimoto et al, 1985; Marnett, 1999).

Bleomycin is administered systemically and it is used only in combination therapy with a number of other antineoplastic agents such as etoposide and cisplatin (Umezawa, 1971; Wharam et al, 1973; Jani et al, 1992ab; Einhorn, 2002). It is most effective against lymphomas, testicular carcinomas, and squamous cell carcinomas of the cervix, head, and neck (Povirk and Austin, 1991; Lazo et al, 1996). Like many other antitumor drugs, bleomycin also manifests clinical limitations. At high doses (>235 mg), bleomycin can induce pulmonary fibrosis, a condition that is triggered by lipid peroxidation (Ekimoto et al, 1985; Wang et al, 2000; Nagase et al, 2002), resulting in pulmonary insufficiency leading to fatal hypoxemia (Sikic, 1986; Harrison and Lazo, 1987; Nagase et al, 2002). Another aspect that limits the clinical application of bleomycin is tumor resistance (Lazo et al, 1996). So far, no definitive mechanism, e.g., drug efflux or enhanced repair of bleomycin-induced DNA lesions or inactivation of bleomycin, explains the development of tumor resistance towards the drug (Miyaki et al, 1975; Akiyama et al, 1981; Sebti et al, 1991; Morris et al, 1992; Urade et al, 1992). Although, current findings from our laboratory raised the possibility that tumor resistance to bleomycin might be at the level of drug uptake, instead.

A. Properties and mechanism of action of bleomycin

Bleomycin consists of three regions that include a carbohydrate moiety, a metal binding domain, and a DNA binding domain (Figure 1). The latter contains a chemical structure similar to the composition of polyamines (Hecht, 1986; Kane et al, 1994; Petering et al, 1996; Abraham, 1999; Leitheiser et al, 2000; Hoehn et al, 2001). Bleomycin also contains at least two accessible amino groups that could form covalent linkages with other compounds including fluorescein, thus providing a way to create a fluorescently labeled form of the drug (see below). Reduce Fe²⁺, as well as other metal ions, can bind to the metal pocket and trigger activation of bleomycin (Burger et al, 1979, 1981). During activation, the reduced Fe²⁺becomes oxidized to Fe³⁺ resulting in the production of free radicals (Burger et al, 1979, 1981). The activated drug can intercalate with DNA to generate at least 4 types of DNA lesions, and the extent of formation of these lesions depends upon the oxygen content of the cell (Umezawa et al, 1966; Suzuki et al, 1970b; Terasima et al, 1970; Burger, 1998; Tounekti et al, 2001). For example, in the absence of oxygen bleomycin abstracts a hydrogen atom from the C4¢ position of the sugar moiety to create an unstable ring. This unstable sugar can ring open to form a mutagenic lesion, apurinic/apyrimidinic (AP) site, where the DNA strand is intact, but it lacks a base (Worth et al, 1993; Burger, 1998). In the presence of oxygen, bleomycin can damage the sugar moiety resulting in a single strand break blocked at the 3¢-terminus with a fragment of the sugar (Giloni et al, 1981; Worth et al, 1993; Burger, 1998). This latter lesion, 3¢-phosphoglycolate, blocks DNA repair synthesis and therefore must be removed in order to promote cell division (Giloni et al, 1981; Worth et al, 1993; Burger, 1998). It is noteworthy that the remaining portion of the fragmented sugar exists in the free base propenal form, which has the ability to react with the DNA to form base adducts. For example, the base propenal bears a malondialdehyde moiety, which can react with guanine to form the most abundant adduct pyrimidopurinone of deoxyguanosine (Dedon et al, 1998). Bleomycin also produces bi-stranded DNA lesions at certain specific sequences, such as CGCC, which are generated when the Fe.bleomycin complex creates an AP site on one strand, and a directly opposed single strand break on the complementary strand (Steighner and Povirk, 1990ab; Dedon and Goldberg, 1992; Absalon et al, 1995; Hoehn et al, 2001). The spontaneous cleavage of the AP site by primary amines (e.g., histone amine) in vivo converts the bi-stranded lesions into a double strand break (Steighner and Povirk, 1990ab; Dedon and Goldberg, 1992; Absalon et al, 1995). In general, bleomycin produces structurally- and chemically-related lesions as those created by ionizing radiation, and therefore it is considered a radiomimetic agent (Steighner and Povirk, 1990ab; Dedon and Goldberg, 1992; Absalon et al, 1995; Hoehn et al, 2001).

Bleomycin-induced DNA lesions are known to be highly mutagenic, and account for its potent antitumor ability (Steighner and Povirk, 1990ab; Bennett et al, 1993; Tates et al, 1994; Dar and Jorgensen, 1995; Pavon et al, 1995). Thus, if tumor cells can rapidly repair bleomycin-induced lesions, then they are likely to become resistant to the genotoxic effects of the drug. Likewise, normal cells of cancer patients exposed to bleomycin must rely on DNA repair enzymes to process effectively the drug-induced lesions, in order to prevent lethal mutations that could lead to secondary tumors.

B. Potential mechanisms leading to cellular bleomycin-resistance

As pointed out above, tumor resistance is a major obstacle to bleomycin chemotherapy (Morris et al, 1991; Sebti et al, 1991; Jani et al, 1992ab). To date, no definitive mechanism(s) is known to explain how tumors acquire such resistance, although possible ones might include (i) enhanced repair of bleomycin-induced DNA lesions, (ii) increased drug efflux by multidrug transporters, (iii) inactivation of the drug by elevated levels of bleomycin hydrolase, and (iv) decreased drug uptake by plasma membrane permeases (Miyaki et al, 1975; Akiyama et al, 1981; Sebti et al, 1991; Morris et al, 1992; Urade et al, 1992; Sanz et al, 2002). These possibilities are discuss below, as well as striking findings from our laboratory indicating that bleomycin-resistance is due principally to altered drug uptake.

1. DNA repair enzymes

Organisms exposed to bleomycin must recruit proteins to repair bleomycin-induced DNA lesions in order to avert the mutagenic effects of the drug (Ramotar and Wang, 2003). Over the years, we employed the yeast Saccharomyces cerevisiae as a model organism to search for enzymes that would repair bleomycin-induced DNA lesions, as such enzymes have not been characterized in mammalian cells (Ramotar, 1997; Sander and Ramotar, 1997; Jilani et al, 1999). S. cerevisiae provided a solid foundation for this study due to its multifaceted advantages that include (i) a powerful genetic system that permits rapid creation of gene nulls, (ii) a wealth of readily available technologies and a vast database information, and (iii) the ability to isolate clinically-relevant human disease genes by cross-species complementation (Phizicky and Fields, 1995; Bassett et al, 1996; Lashkari et al, 1997; Andrade et al, 1998; Pereira, 1998; Neff et al, 1999; Steinmetz and Davis, 2000; Steinmetz et al, 2002).

To date, we and others have biochemically characterized three enzymes, i.e., Apn1, Apn2, and Tpp1, that clearly act in vitro to directly process bleomycin-induced DNA lesions (Ramotar et al, 1991; Ramotar, 1997; Sander and Ramotar, 1997; Jilani et al, 1999; Vance and Wilson, 2001; Jilani and Ramotar, 2002). Genetic studies revealed that mutants lacking all three enzymes displayed severe hypersensitivity to bleomycin, while single mutants showed virtually no sensitivity to the drug, as compared to the parent (Ramotar, 1997; Vance and Wilson, 2001). These findings clearly indicate that more than one of these enzymes can compete to repair bleomycin-induced lesions in vivo (Ramotar et al, 1991; Ramotar, 1997; Sander and Ramotar, 1997; Jilani et al, 1999; Vance and Wilson, 2001; Jilani and Ramotar, 2002). Despite extensive searches, the Apn1 homologue has not been found yet in humans, although the counterparts of yeast Apn2 (hApe/ref-1) and Tpp1 (hPNKP) have been identified (Barzilay and Hickson, 1995; Jilani et al, 1999). Recent studies demonstrated that overproduction of hApe1/ref-1 in the testicular cancer cell line NT2/D1 resulted in ~3-fold increased protection against bleomycin(Robertson et al, 2001). However, it remains to be shown if hApe1 overexpression can account for the nearly 15% of patients that resist bleomycin-therapy (Robertson et al, 2001). Whether hApe or hPNKP, and other yet unidentified human DNA repair enzymes (e.g., human Apn1), plays a role in bleomycin-tumor resistance is currently under investigation.

2. Evidence against multidrug transporters as a mechanism of bleomycin-resistance

In mammalian cells, elevated levels of plasma membrane ABC transporters, such as the multidrug resistant efflux pump, MDR1, and the multidrug resistant-associated protein, MRP1, are known to increase efflux of chemotherapeutic agents thereby allowing tumor (and normal) cells to evade drug-induced cytoxicity (Gottesman et al, 1995; Dean et al, 2001). So far, there is no convincing evidence for the involvement of either MDR or MRP efflux pumps in bleomycin resistance (Chen et al, 1994). Likewise in yeast, several well studied ABC transporters showed no involvement in the efflux of bleomycin(Ramotar and Masson, 1996; Decottignies and Goffeau, 1997). Consistent with this observation is that a yeast mutant strain, AD1-8, lacking seven of these transporters (Pdr5, Pdr10, Pdr11, Pdr15, Snq2, Yor1, and Ycf1), some of which share significant similarities to human ABC transporters and are involved in drug efflux, showed high level of sensitivities to numerous toxic compounds (Decottignies et al, 1998; Rogers et al, 2001). However, this mutant strain showed normal parental resistance to bleomycin (DR., unpublished). In other studies, the overexpression of each of these pumps did not confer upon parental yeast strains any additional resistance to bleomycin (DR., unpublished). Thus, it is unlikely that drug efflux pumps play a major role in bleomycin resistance.

3. Bleomycin-hydrolase confers no drug resistance to yeast

It is demonstrated that bleomycin can be metabolically inactivated in normal and tumor tissues by an enzyme called bleomycin hydrolase (Blh1), and that such inactivation may play a role in bleomycin resistance (Umezawa et al, 1974; Sebti and Lazo, 1988; Nishimura et al, 1989; Schwartz et al, 1999). The characterized enzyme acts as a thiol protease and hydrolyzes the b-aminoalanine amide moiety near the DNA binding domain of bleomycin to generate the inactive deamido metabolite (Akiyama et al, 1981; Lazo and Humphreys, 1983; Sebti et al, 1991). The activity can be inhibited by the thiol protease specific inhibitor (E64), and mammalian cells exposed to E64 displayed sensitivity to bleomycin (Jani et al, 1992ab). These observations quickly led to the isolation of the corresponding bleomycin hydrolase gene from yeast and mammalian cells (Enenkel and Wolf, 1993; Magdolen et al, 1993; Bromme et al, 1996; O'Farrell et al, 1999; Ferrando et al, 1996). Expression of the yeast bleomycin hydrolase gene BLH1 in mouse NIH3T3 cells conferred a nearly 5-fold increase resistance to bleomycin, which could be blocked by E64 inhibitor (Pei et al, 1995). Notwithstanding this finding, independent studies showed conflicting data regarding the role of yeast Blh1 in the inactivation of bleomycin (Enenkel and Wolf, 1993; Magdolen et al, 1993 Kambouris, et al. 1992). While two studies showed that blh1D mutants are mildly sensitive to bleomycin, another study clearly established that these mutants exhibit no more sensitivity to the drug than parent strains (Kambouris et al, 1992; Enenkel and Wolf, 1993; Magdolen et al, 1993). In fact, we also demonstrated that blh1D mutants are not sensitive to bleomycin, and that overproduction of Blh1 in yeast cells does not protect bleomycin-hypersensitive mutants from the genotoxic effects of the drug (Wang and Ramotar, 2002). As such, we conclude that in vivo yeast Blh1 has no direct role in mediating cellular resistance to bleomycin, and that the role of the enzyme in producing tumor resistance remains unclear. However, it is worthy of noting that the yeast Blh1 protein, also called Gal6, is under the control of the Gal4 transcriptional activator (Zheng et al, 1997). Blh1/Gal6 binds specifically to the Gal4 transcription factor DNA binding site and acts as a repressor in a manner that negatively controls the pathway of galactose metabolism (Xu and Johnston, 1994; Joshua-Tor et al, 1995; Zheng et al, 1997). Equipped with this new function, it would appear that bleomycin hydrolase plays a more general role by degrading certain transcription factors in order to regulate gene expression, as well as ribosomal proteins (Zheng et al, 1998; Koldamova et al, 1999). If this is the case, the bleomycin resistance observed by expression of yeast Blh1 protein in mammalian cells could be explained, for example, by degradation of pro-apoptotic factors, thus preventing cell death.

4. Iron transport plays no role in bleomycin resistance

So far, we found no evidence in yeast that altered iron metabolism leads to bleomycin resistance. For example, fre1D and fre2D mutants defective in the Fe/Cu reductase genes showed normal parental resistance to bleomycin (Georgatsou and Alexandraki, 1999).

5. Evidence for bleomycin transport across the plasma membrane

Previous evidence suggests that a protein exists on the plasma membrane of mammalian and yeast cells, which is believed to bind bleomycin and mediate its transport into the cell (Poddevin et al, 1991; Aouida et al, 2003). This uncharacterized putative protein has been identified by analyzing plasma membrane fractions, under non-denaturing gel conditions, for specific binding to bleomycin carrying labeled [⁵⁷Co] cobalt in the metal ion pocket (Pron et al, 1993). This preliminary observation prompted us to examine the kinetic parameters of bleomycin uptake into yeast cells. However, this could not be readily achieved with the labeled [⁵⁷Co]-bleomycin and instead we created a fluorescently labeled form of bleomycin to conduct the study (Mistry et al, 1992). The fluorescien conjugated bleomycin (F-bleomycin) has been purified and rigorously shown to retain nearly full capacity to act as a genotoxic agent, as well as to induce cell killing by creating endogenous DNA lesions analogous to the unmodified drug (Aouida et al, 2004a). We then used the F-bleomycin as a tool to show that the drug can be actively transported into parent strains in a concentration- and time-dependent manner (Aouida et al, 2004a). Moreover, these studies revealed that F-bleomycin transport may be dependent upon new protein synthesis, as uptake could be blocked by the protein synthesis inhibitor cycloheximide (Aouida et al, 2004a).

Base on the above observations, we reasoned that a plasma membrane transporter must exist to permit bleomycin entry into the cell. If this is the case, mutants devoid of the transporter are expected to exhibit greater than parental resistance to bleomycin. In support of this prediction, a recent report documented that the copper transporter, Ctr1, is responsible for transporting the anticancer drug cisplatin into yeast and mammalian cells (Ishida et al, 2002). Yeast mutants lacking the Ctr1 transporter are resistant to cisplatin, and the overexpression of Ctr1 has been shown to cause increased sensitivity to the drug (Ishida et al, 2002). However, neither the Ctr1 nor other metal ion transporters in yeast is involved in bleomycin transport, favoring the possibility that the transporter in question is likely to exist in nature.

6. Bleomycin accumulates in the vacuoles following transport

With the aid of fluorescent microscopy, we have shown that following F-bleomycin uptake into the parent strain the drug accumulated into the vacuoles (Aouida et al, 2004a). Since the vacuoles serve a function to degrade many macromolecules, it is reasonable to assume that bleomycin might be detoxified in this organelle. Interruption of the endocytotic pathway to the vacuoles caused F-bleomycin to be redistributed such that it is accumulated in the cytoplasm, where the drug can now readily diffuse into the nucleus and rapidly degrade the DNA (Aouida et al, 2004a). In general, all the mutants tested so far with defects in the endocytic pathway to the vacuole display marked hypersensitivity to bleomycin (Table 1). Thus, it would seem that bleomycin is actively transported across yeast plasma membrane and directed to the vacuole, where this organelle might serve as a first line of defense by containing the drug and preventing its cytotoxicity and genotoxicity.

To date, several approaches have been assessed to improve the genotoxicity of bleomycin that include exploiting chemical modifications, as well as directly transferring the drug into the cell via electroporation in order to bypass the vacuoles (Aouida et al, 2003).Although the latter technique seems promising in that it allows a defined number of bleomycin molecules into the cell and causes enhanced bleomycin cytotoxicity, this approach remains cumbersome (Orlowski et al, 1988; Kotnik et al, 2000). However, a more recent study employed the use of photochemical damage to the endocytic vesicles in order to trigger disruption of the organelles, with concomitant release of the accumulated bleomycin, in various cancer cells challenged with the drug (Berg et al, 2005). While this approach holds great promise to enhance the chemotherapeutic effects of the bleomycin (Berg et al, 2005), other rationale approaches are still forthcoming.

C. Genome-wide screen

In the last five years, several genome-wide screens have been performed with different collections of yeast mutant cells exposed to various DNA damaging agents including g-rays, ultraviolet radiation, methyl methane sulfonate, and cisplatin (Bennett et al, 2001; Birrell et al, 2001; Chang et al, 2002; Desmoucelles et al, 2002; Giaever et al, 2002; Fry et al, 2005). These screens have allowed the identification of many previously reported gene functions, as well as uncovered new ones that are required to protect cells against DNA damaging agents, and which otherwise could not be rapidly isolated by other strategies that include the use of transposon-insertion mutagenesis and reverse genetics (Burns et al, 1994). The data generated from these genome-wide screens are being exploited to further establish, for example, those genes encoding proteins that participate in related biological processes (Fry et al, 2005). As discussed below, we have undertaken a similar genome-wide approach to unravel the gene functions that are involved in protecting cells against the genotoxic effects of bleomycin.

1. Bleomycin-hypersensitive mutants revealed by a genome-wide screen in yeast

To directly complement our previous efforts to understand how cells provide resistance to bleomycin, we performed two independent robot-aided screens of the entire collection of haploid yeast mutants to identify all the bleomycin-hypersensitive mutants (Aouida et al, 2004b). The collection comprises single deletions in nearly 4,000 of the 6,000 yeast genes (www.uni-frankfurt.de/fb15/mikro/euroscarf/)(Winzeler et al, 1999; Desmoucelles et al, 2002): The remaining 2,000 genes are essential for cell viability. The screens reproducibly identified 231 mutants that have been independently confirmed, and showing hypersensitivity to bleomycin (i.e., 4- to 20-fold more sensitive than the parent strain). Table 1 list all the genes deleted in the corresponding mutants. Among these genes, we previously identified

Table 1. BLM-hypersensitive and–resistant genes

DNA repair Chromatin Structure	Cell Cycle	Mitochondria and ATP metabolism	Vacuoles and vesicular transport	Cell wall	Unknown Function
	CDC50* ^a			ANP1	Unknown Function
	CTK2			CAX4 ^b	APQ13
CTF18* ^a	DOC1 ^b	ADK1* ^b	AKR1 ^b	CHS1	BAT2
CTF4* ^{a + b}	HTL1 ^{a + b}	AFG3	APG17	CWH36 ^b	GON7
CTF8 ^{a + b}	PHO85*	ATP11	APL2	FKS1	SWF5
EST2*	RTS1*	ATP12	CHC1*	FYV6	YBR168W
FAB1*	SFP1 ^b	ATP14	DID4*	GAS1	YBR267W*
IWR1* ^b		ATP15	END3*	HOC1	YDR049W*
MRE11* ^a	RNA	CAT5*	GLO3	LAG2	YDR532C
NAT3* ^{a + b}	Metabolism	ILM1	IES6	MNN9	YGL072C
RAD27 ^a	ARC1*	ISA1*	INP53*	MNN10	YGR237C
RAD54* ^a	BRE5*	MDJ1	LCB4*	OST4	YGR272C
RAD57* ^{a + b}	BRF1	MRPL51*	LUV1	RMD7	YLR374C
RAD6* ^{a + b}	CDC40* ^{a + b}	MRPS8	MON2*	ROT2*	YMR031W-A^a
RAI1* ^b	DBP7	MSF1	PEP5*	SLG1*	YOR342C
REM50 ^a	DIA4 ^b	MSY1*	PEP12*		YPR044C
RNR1	KEM1*	NHX1	RIC1
RNR4*	LOC1 ^b	OCT1	RCY1*	Miscellaneous
SNF6	PAT1* ^b	PDA1	RVS161* ^b	ADE12
SPT10 ^b	SAC3*	PFK2 ^b	RVS167 ^b	ADH1
SPT20	SNT309	PPA2	SHE4	APM1	BLM-
THP1*	YER087W*	RML2	SWF1*	ARP5*	resistant
TRF4*		RSM19	VAM6	CYS4	AGP2*
VID31* ^{a + b}	Protein	RSM7 ^b	VPH2 ^b	DIA2	FES1*
XRS2 ^{a + b}	Synthesis	RSM22	VPS1	GLY1	PTK2*
	ASC1	SNF1	VPS3	GON1*	SKY1*
	EAP1	SPF1*	VPS4	GPH1	BRP1
Transcription	EGD2	SSQ1	VPS8	GUP1
ASF1* ^{a + b}	PDR13 ^b	SWF3	VPS9	MET22*
BUR2 ^{a + b}	PFD1 ^b	SWS2	VPS15	NPL6^a
CCR4 ^b	RPL13B	TOM5	VPS16*	OPT2
CTK1	RPL1B	TUF1*	VPS20	PLC1
CTK3	RPL27A	UGO1*	VPS24	PMP3
DHH1* ^b	RPL35A	YDJ1	VPS25*	PRO1
GAL11	RPL39	YHM1	VPS27	REG1
IMP2	RPS0B*	YME1	VPS45*	RIB4
KCS1	RPP1A		VPS66	SHP1
POP2	TIF3	Polarity and polarized growth	VPS67	SLX8*
ROX3	TIF4631	BEM2	VPS69	SPS4
RPB4	ZUO1* ^b	BUD16*	YAF9*	TPS1
RPB9* ^{a + b}	YIF2* ^b	BUD20*	YPT6
RRN10 ^a		BUD23*	YPT7
SIN4	Protein Degradation	BUD25^a
SPT21	GRR1* ^{a + b}	BUD27	Cytoskeleton
SPT7*	UBP3	BUD31*	ARC18
SRB2 ^a	UMP1*	BUD32*	CDC10*		^aMMS sensitive
SRB5 ^a		SAC2	CNM67 ^b
SRB8*		SAC6 ^b	GCS1		^bIR sensitive
SSN8	Lipid metabolism		HOF1* ^{a + b}		* 88 genes conserved in human
SWI4	ARV1*		SPC72
SWI6* ^a	ERG2
TAF14*	ERG4

IMP2, GAS1, SLG1, END3, and RAD6 by a different type of screen namely insertional-mutagenesis that causes functional disruption of genes (He et al, 1996; Masson and Ramotar, 1996; Leduc et al, 2003), thus validating the utility of our genome-wide approach. From the collection of genes, 88 encode proteins that share significant level of identity with a human protein (Table 1, shown with an asterisk), suggesting that yeast and human cells may conserve the same biological processes to combat the cytotoxic and genotoxic effects of bleomycin.

The genes (Table 1) encode proteins belonging to several functional groups including DNA repair and chromatin structure, transcription, and cell cycle. Other groups participate in maintaining the vacuolar and mitochondrial functions. In fact, the largest number of genes identified belongs to the vacuolar pathway. Thus, it would appear that modulating the function of these gene products could likely affect how cancer cells respond to bleomycin. For example, blocking the detoxification pathway might effectively increase the cytoplasmic concentration of bleomycin (Aouida et al, 2004a; Berg et al, 2005). In addition, many chromatin-remodeling proteins such as Snf6, Spt20, and Spt10 might play a role in promoting specific repair of bleomycin-induced DNA lesions. These latter chromatin-remodeling proteins are under characterization in order to examine for relationships with DNA repair pathways and to determine the types of DNA lesions that are processed.

2. Bleomycin-resistant mutants revealed by the genome-wide screen in yeast

In addition to the search for bleomycin-hypersensitive mutants, we also exploited the screen to identify those mutants that would be resistant to the drug. At least five mutants that displayed nearly 500- to 3000-fold more resistance to bleomycin than the parent have been identified (Table 1) (Aouida et al, 2004b). The mutants lacked the gene AGP2, PTK2, SKY1, FES1, and BRP1 (Table 1). Amongst these mutants, the agp2D mutants exhibited the greatest resistance (~3000-fold) to bleomycin, while the other four displayed at least 500-fold more resistance than the parent (Aouida et al, 2004b). Below, we highlighted the current knowledge of the five gene products involved in bleomycin-resistance.

i. Agp2

Agp2 is a 67.2-kDa plasma membrane protein that belongs to the amino acid transporter family (Lee et al, 2002; Schreve and Garrett, 2004). Previous studies demonstrated that Agp2 is involved in the uptake of L-carnitine, which serves as a carrier to transport the end product of fatty acid b-oxidation, acetyl-CoA, from the peroxisome into the mitochondria for complete oxidation (van Roermund et al, 1999; Lee et al, 2002). However, it remains unclear if Agp2 has a direct role in L-carnitine transport, although there is a correlation showing that the expression level of the transporter is induced when cells are grown in the presence of fatty acid (van Roermund et al, 1999). We recently showed that Agp2 is responsible also for mediating the uptake of bleomycin into the cells (Aouida et al, 2004b). This observation is derived from the finding that agp2D mutants are unable to transport F-bleomycin into the cells causing a decreased accumulation in the vacuoles (Aouida et al, 2004b). However, reintroduction of the AGP2 gene into the agp2D reinstated F-bleomycin uptake and its accumulation into the vacuoles (Aouida et al, 2004b). If the Agp2 transporter is overproduced via increased gene dosage, it can greatly enhance the uptake of F-bleomycin and simultaneously sensitized the cells to killing by the drug, a consequence of increased damage to the chromosomal DNA (Aouida et al, 2004b). The overproduced Agp2 did not sensitize cells to various other DNA damaging agents including cisplatin, suggesting that Agp2 is selectively involved in bleomycin uptake (Aouida et al, 2004b). We reasoned that Agp2 is involved in the uptake of both bleomycin and L-carnitine as the bleomycin specie (bleomycin-A5) we used in the genome-wide screen possesses a region with a chemical structure that is similar to L-carnitine and polyamine (Figure 1). In fact, preincubation of cells with L-carnitine blocked the uptake of bleomycin and protected the cells from bleomycin-induced cell death, further supporting the notion that Agp2 functions to transport the bleomycin-A5 specie (Aouida et al, 2004b). It is noteworthy that agp2D mutants showed only modest resistance to another specie of bleomycin, that is bleomycin-A2, which lacks the polyamine portion (M.A. and D.R., unpublished), indicating that this region plays a critical role in permitting entry of the drug into the cell. This observation prompted us to examine if the Agp2 transporter might in fact be the long sought high affinity polyamine transporter, particularly since a transporter of this nature remains unidentified in higher eukaryotic cells. Uptake studies performed with labeled spermidine revealed that Agp2 is indeed the high-affinity polyamine permease in yeast (Aouida et al, 2005). Deletion of the AGP2 gene dramatically reduces the initial velocity of spermidine and putrescine uptake and confers strong resistance to the toxicity of exogenous polyamines (Aouida et al, 2005). Reintroduction of the AGP2 gene from an expression vector restored polyamine transport into the agp2D mutants (Aouida et al, 2005). Further analyses revealed that the transporter function of Agp2 is crucial to sustain the growth of cells (e.g., spe1D mutant) lacking the ability to synthesize polyamine (Aouida et al, 2005). In fact, spe1D agp2D double mutants required more than 10-fold higher concentrations of exogenous putrescine to restore cell proliferation, as compared to the spe1D single mutant (Aouida et al, 2005). Other genetic alterations, such as disruption of the END3 gene encoding a protein required for an early step of endocytosis, increase the abundance of Agp2 and causing a marked upregulation of spermidine transport velocity (Aouida et al, 2005). Collectively, these observations are consistent with the notion that (1) Agp2 is the first eukaryotic permease that preferentially uses spermidine over putrescine as a high-affinity substrate, and (2) Agp2 plays a central role in the uptake of polyamines into yeast cells. As such, we expect that agp2D mutants would be resistant to other chemotherapeutic agents conjugated to polyamine, e.g., chlorambucil-spermidine and difluoropolyamines (Hull et al, 1988; Cullis et al, 1994).

ii. Ptk2

Ptk2 is a Ser/Thr protein kinase that belongs to the Npr1 kinase family, and which regulates the activity of permeases and transporters (Schmidt et al, 1998). We previously showed that Ptk2 is a key regulator of polyamine transport into yeast cells (Kaouass et al, 1997). Genetic studies revealed that ptk2D mutants are unable to transport polyamine into the cells and thus these mutants are hyperresistant to toxic doses of spermine (Kaouass et al, 1997). Ptk2 has been shown to play a role in positively regulating the activity of the plasma membrane proton pump Pma1, which creates a voltage gradient that serves to provide energy for transporters (Goossens et al, 2000). Whether Ptk2 is required to directly phosphorylates Pma1 remains to be seen, but phosphorylation of Ser899 activates Pma1 activity (Portillo, 2000). In this context, we postulate that the voltage gradient created by Pma1 is also required to propel the function of the Agp2 transporter. Thus, it is logical to expect that mutants defective in Ptk2 function would display resistance to bleomycin and polyamines. As predicted from the functions of Ptk2 and Agp2, it is not surprising that agp2D ptk2D double mutant exhibits the same level of bleomycin and polyamine resistance as the single agp2D mutant (unpublished data). From the above findings, it would appear that mammalian cells compromised for the maintenance of the voltage gradient is likely to display resistance to bleomycin.

iii. Sky1

Sky1 is a Ser/Arg kinase also shown to regulate polyamine transport (Erez and Kahana, 2001), and that sky1D mutants are hyperresistant to toxic doses of spermine (Erez and Kahana, 2001). Since the emerging scenario in yeast is that a family of protein kinases exists which is dedicated to regulate plasma membrane transporter activity, it is possible that Sky1 could control Agp2 activity by phosphorylation (Schmidt et al, 1998). However, neither Agp2 nor the other proteins involved in bleomycin resistance, possesses a Ser/Arg rich domain that can serve as a phosphorylation acceptor site for the action of Sky1 kinase. Alternatively, Sky1 could affect the phosphorylation status of proteins with Ser/Arg rich domain that are involved in RNA binding activity and cellular location, and thus indirectly affecting the function of Agp2 (Gilbert et al, 2001).

In the case of mammalian cells, a dominant negative form of the mammalian Sky1 kinase (SRPK1 serine kinase) has been shown to confer upon Chinese hamster lung fibroblast and HeLa cells resistance to bleomycin, but not to other DNA damaging agents (Sanz et al, 2002). This observation strongly suggests that a conserved pathway exists in eukaryotic cells to regulate the genotoxic effects of bleomycin (Sanz et al, 2002; Aouida et al, 2004b). Thus, unraveling the mechanism by which the dominant negative SRPK1 triggers bleomycin-resistance could be an important clue to understand the nature of this drug resistant pathway and implement better regimens.

iv. Fes1

Fes1 is associated with the ribosomes and believed to play a role in protein translation (Kabani et al, 2002). fes1D mutant is believed to have a translational defect, suggesting that Fes1 could signal translation of at least one of the components (Agp2, Sky1, Ptk2, and Brp1) involved in bleomycin resistance. So far, we found no evidence that the expression level of a functionally tagged form of Agp2, Agp2-cTAP (cTAP carrying protein domains for Tandem Affinity Purification), is diminished in the fes1D mutant. Clearly, additional studies are needed to precisely determine the function of Fes1, and if it is involved in promoting the translation of the other members.

v. Brp1

We designated the YGL007w gene as BRP1, which is predicted to encode a small 13-kDa bleomycin resistant protein of unknown function. Since the activity of some membrane transporters is regulated by small proteins, e.g., the small heat shock protein Hsp30 regulates the activity of the H⁺/ATPase, Pma1, it is possible that Brp1 might engage in a similar function (Braley and Piper, 1997; de la Fuente et al, 1997). Consistent with this notion, one group, which quickly reproduced our findings, provided preliminary evidence that Brp1 might be required to regulate the expression level of Pma1 (Porat et al, 2005).

D. Model

It is clear from our studies that deletion of any of the following five yeast genes, AGP2, PTK2, SKY1, FES1, and BRP1 protects the organism against the cytotoxic and genotoxic effects of bleomycin (Aouida et al, 2004b). Thus, to explain the possible roles played by these gene products in bleomycin resistance, we propose the following model (Figure 2) that is most consistent with the current data. The Agp2 protein acts as the transporter of bleomycin and that its activity is likely regulated directly by the Sky1 kinase. In this model, the Ptk2 kinase acts to modulate the proton pumping activity of Pma1 to provide a voltage gradient needed to drive the activity of many transporters including Agp2. In contrast, the roles of Fes1 and Brp1 are less clear. However, we speculate that Fes1 could control the translation of either Sky1 or Ptk2 or Brp1, and that this latter protein in turn regulates the expression level of the proton pump Pma1. We believe that in depth functional analysis of these gene products will provide a much better understanding of the mechanism by which bleomycin enters the cell, and will lead to the identification of novel therapeutic targets aimed at enhancing the antitumor properties of the drug.

II. Conclusions

To date, a homologue of Agp2 has not yet been reported in humans, but two high affinity L-carnitine transporters CT2 and OCTN2, each sharing approximately 19% identity, have been identified (Tamai et al, 1998; Enomoto et al, 2002). Interestingly, hCT2 is expressedexclusively in human testis, whereas OCTN2 is expressed stronglyin kidney, skeletal muscle, heart, and prostate (Tamai et al, 1998; Enomoto et al, 2002).The fact that hCT2 is expressed exclusively in the testis and thattesticular cancers have a high cure rate with bleomycin therapy isstriking, offering strong support for the notion that hCT2 couldbe the human transporter of bleomycin. If this is the case, hCT2 could play an important role in predicting the responses of patients to bleomycin. For example, mutations altering hCT2 transporter function are expected to be an important factor in tumors that gradually develop resistance to bleomycin. Extensive studies are in progress to examine if hCT2 is involved in both bleomycin and polyamine uptake (M.A and D.R., in preparation). We believe that seeking strategies to upregulate the transporter activity can lead to widening the application of bleomycin therapy to other cancers besides testicular.

Acknowledgements

The work described in this review is supported by a grant (MT-121391) to D.R. from the Canadian Institutes of Health Research. D.R. is awarded a senior fellowship from the Fonds de la Recherche en Santé du Québec. M.A. is awarded a post-doctoral fellowship from the National Cancer Institute of Canada.

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Dindial Ramotar

Review Article

Received: 03 February 2006; Revised: 17 April 2006

Accepted: 5 May 2006; electronically published: May 2006

3. Bleomycin-hydrolase confers no drug resistance to yeast

5. Evidence for bleomycin transport across the plasma membrane

SWF5

ARC1*

SPS4

BUD16*

TPS1

BUD20*

CDC10*

CNM67 ^b

Acknowledgements

References

Review Article

Received: 03 February 2006; Revised: 17 April 2006

Accepted: 5 May 2006; electronically published: May 2006

3. Bleomycin-hydrolase confers no drug resistance to yeast

5. Evidence for bleomycin transport across the plasma membrane

SWF5

ARC1*

SPS4

BUD16*

TPS1

BUD20*

CDC10*

CNM67 b

Acknowledgements

References

CNM67 ^b