DNA interstrand cross-link repair: understanding role of Fanconi anemia pathway and therapeutic implications

REVIEW ARTICLE

DNA interstrand cross-link repair: understanding role ofFanconi anemia pathway and therapeutic implicationsPallavi Shukla, Avani Solanki, Kanjaksha Ghosh, Babu Rao Vundinti

Department of Cytogenetics, National Institute of Immunohaematology (NIIH), Mumbai, India

Abstract

Interstrand cross-links (ICLs) are extremely toxic DNA lesions that prevent DNA double-helix separation

due to the irreversible covalent linkage binding of some agents on DNA strands. Agents that induce these

ICLs are thus widely used as chemotherapeutic drugs but may also lead to tumor growth. Fanconi anemia

(FA) is a rare genetic disorder that leads to ICL sensitivity. This review provides update on current

understanding of the role of FA proteins in repairing ICLs at various stages of cell cycle. We also discuss

link between DNA cross-link genotoxicity caused by aldehydes in FA pathway. Besides this, we

summarize various ICL agents that act as drugs to treat different types of tumors and highlight strategies

for modulating ICL sensitivity for therapeutic interventions that may be helpful in controlling cancer and

life-threatening disease, FA.

Key words interstrand cross-links; Fanconi anemia; cancer; chemotherapy

Correspondence Babu Rao Vundinti, National Institute of Immunohaematology (NIIH), 13th floor, New Multistoreyed Bldg,

KEM Hospital Campus, Parel, Mumbai 400012, India. Tel: +9122 24138518, 24138519, 24111161; Fax: +9122 24138521;

e-mail: [email protected]

Accepted for publication 8 July 2013 doi:10.1111/ejh.12169

Interstrand cross-links (ICLs), highly toxic DNA lesionsbetween the two complementary strands of the doublehelix, require network of complicated DNA repair pro-cesses for their removal (1). Among these repair pro-cesses, proteins of Fanconi anemia (FA) pathway play animportant role in ICL repair. Many ICL-inducing drugssuch as mitomycin C, psoralens, nitrogen mustards, andplatinum compounds are widely used as chemotherapeuticdrugs against leukemia and solid cancers. These drugsfunction by inducing interstrand cross-links by bindingthrough covalent linkage on DNA strands that is irrevers-ible. This leads to inhibition of DNA replication and tran-scription and results in structural changes in DNA leadingto altered cellular processes, for example, nitrogen mus-tards and platinum compounds act by reacting with basesat each DNA strand at N-7 position of guanosine or aden-osine (2). Mitomycin C and psoralens bind to DNA bycycloaddition or cycloreduction of their planar rings (3).Although the DNA cross-linking drugs are frequently usedas anticancer drugs, exposure to high levels of these drugsmay lead to cancer. This is because these agents can

cause wide variety of DNA lesions in addition tocross-links, for example, cisplatin can cause 90% ICLs inaddition to 5% ICLs, while nitrogen mustards only cause5–10% ICLs. Thus, repair mechanisms of lesions causedby ICLs may cause point mutations as well as deletionsand translocations leading to tumor growth (3).The ICL repair mechanism has not yet been fully under-stood. FA is a good model to understand the molecularevents involved in carcinogenesis and chemotherapeuticeffect of ICLs as it exhibits sensitivity to ICL-inducingagents. Moreover, defective FA proteins are known to beinvolved in various types of cancers. Here, we discuss therole of FA proteins in repairing ICLs as well as discussvarious ICL-based chemotherapy. We also highlight varioustherapeutic strategies for modulating ICL sensitivity incontrolling tumors and FA.

Fanconi anemia

Fanconi anemia is an autosomal recessive disorder (OMIM227650) and is characterized by bone marrow failure (aplastic

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 381

European Journal of Haematology 91 (381–393)

anemia), developmental delay, physical abnormalities, andincreased incidence of cancer (4, 5). To date, at least 15 genesbelonging to FA complementation groups, A, B, C, D1(BRCA2), D2, E, F, G, I, J (BRIP1), L, M, N (PALB2), O(RAD51C), and P (SLX4), are known. A part from thesegenes, other genes DDX11 and FAAP20, are thought as tenta-tive to belong to FA complementation group (6). Mutation inany one of these genes can lead to FA. The majority ofreported cases of FA are due to mutations in FANCA (65%),FANCC (15%), or FANCG (10%) (7). Cells derived frompatients with FA are hypersensitive to DNA cross-linkingagents and may use alternative pathways of repair that lead todeleterious genetic aberrations such as radial chromosomes.Alterations in the FA pathway proteins have been reported invarious types of cancers such as breast cancer, ovarian cancer,lung cancer, cervical cancer, and pancreatic cancer(8–12).Many patients with FA develop AML, solid tumors with themost common being head and neck squamous cell carcinoma(HNSCC). The failure to repair ICLs and the consequenttumorigenesis in individuals with FA indicate that the FAgenes are essential for an ICL repair pathway. FA genes,including the BRCA2 (also known as FANCD1) and breastand ovarian cancer tumor suppressor, participate in a commonpathway of ICL repair. Aldehydes, which are carcinogenic,have been shown to directly modify bases in vitro, can lead toDNA–protein and DNA–DNA cross-links, and damage DNA.Endogenously, aldehydes are produced as by-products ofseveral metabolic pathways, such as lipid peroxidation, carbo-hydrate or metabolism ascorbate autoxidation, amine oxidases,cytochrome P-450s, or myeloperoxidase-catalyzed metabolicactivation (13). Several studies have reported that cellsexposed to acetaldehyde accumulate DNA damage (14, 15).Ethanol exposure (a dietary source of acetaldehyde) results inDNA damage in mice and humans (16). Studies have demon-strated chicken DT40 DNA repair mutant cell lines(ΔFANCB, ΔFANCC, ΔFANCJ, and ΔRAD51C) for hyper-sensitivity to formaldehyde. (17). Hence, FA pathway genesseem to be specifically required for cellular resistance to acet-aldehyde. It has been demonstrated that the FA DNA repairpathway counteracts acetaldehyde-induced genotoxicity inmice as the acetaldehyde-catabolizing enzyme Aldh2 is essen-tial for the development of Fancd2�/� embryos (17). Com-bined inactivation of aldehyde catabolism and the FA DNArepair pathway in mice leads to development of aplastic ane-mia with concomitant accumulation of damaged DNA withinthe hematopoietic stem and progenitor cell (HSPC) pool (18).This suggests that aldehyde genotoxicity affects mostly toHSPC cell population and explains why progressive loss ofthe HSPC pool leads to development of bone marrow failurein most patients with FA. In response to replicative stress andunresolved DNA damage, p53 is hyperactivated in FA cellsand triggers a late p21Cdkn1a-dependent G0/G1 cell cycle arrestto limit deleterious DNA damage accumulation and preservethe genome integrity (19).

Role of FA pathway proteins in ICL repair at

different stages of cell cycle

Fanconi anemia proteins along with different nucleases andpolymerases play their role for repairing diverse DNA cross-links at various stages of cell cycle (Fig. 1). The majority ofFanconi anemia proteins take part in ICL repair in S phaseof the cell cycle.

G1 phase

In G1 phase of the cell cycle, ICLs are removed by nucleo-tide excision repair (NER). Endonucleases XPF-ERCC1,specific for 3′ flap structures, which, combined by 5′ flapactivity of Fanconi-associated nuclease 1 (FAN1), couldcleave DNA backbone on both sides of an ICL resulting indistortion of the DNA double helix (3). This is followed bytranslesion synthesis and excision of ‘flipped out’ ICLs.SLX4 (FANCP) could also bind to distorting ICLs in thisphase by interacting with the mismatch repair (MMR)complex MutSß directly (20).However, only a subset of ICLs is repaired in G1 phase

and some lesions can be bypassed as they undergo futilecycle of excision and repair in this manner. These unpro-cessed ICL then proceed to S phase of the cell cycle to getrepaired by homologous recombination events.

Mid/early S phase

Eight of the FA proteins (FANCA, FANCB, FANCC,FANCE, FANCF, FANCG, FANCL, FANCM) are knownto form FA core complex (21). ICLs that are induced duringS phase of the cell cycle activate FA core complex to mono-ubiquitinate two DNA-associated protein, FANCD2 andFANCI (22). This FA core complex activation is mediatedby FANCL, a catalytic E3 ubiquitin ligase subunit requiredfor the monoubiquitylation of FANCD2 (23). Absence ordefect in any one of the proteins of FA core complex willlead to failure of ubiquitination resulting in unrepaired DNAdamage. During the S phase, when the damage is encoun-tered by replication fork, replication stalls due to the cova-lent linkage between the two DNA strands. This damage isthen recognized by FANCM, which contains two domains,ERCC4 nuclease domain that binds to branched DNA struc-ture and an internal domain that interacts with FA corecomplex. FANCM interacts with another ERCC4 domain-containing protein, FAAP24, to form a FANCM–FAAP24heterodimer, which helps in recruitment of the FA core com-plex to chromatin. During S phase, a partially processedintermediate may be encountered by a replication fork,which leads to collapse of the replication fork. This collapseof the replication fork is probably avoided by FANCM-med-iated stabilization of the fork. The encounter of ICL by thereplication fork leads to nuclease recruitment at the site of

382 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

DNA interstrand cross-link repair in FA Shukla et al.

damage. Two proteins, FAN1 and DNA polymerase v (Polv or POLN), are known to colocalize with modified form ofFANCD2 in nuclear foci during DNA damage (24). FAN1has a UBZ4-type ubiquitin-binding zinc finger domain,which is required for interaction with modified FANCD2.SLX4 could be recruited directly to the vicinity of DNAdamage by its tandem UBZ domains. Finally, MUS81 andXPF are localized as part of SLX4 complex. FAN1 degradessingle-stranded DNA (ssDNA) or double-stranded DNA(dsDNA) exposed ends and can cut DNA on the 5′ side of assDNA–dsDNA junction and thus possess activity that cancut DNA that is adjacent to stalled replication fork. Suchcleavage is called ‘unhooking’ and can convert the stalledfork into a double-strand break (DSB). Unhooking of theICL by coordinated incision by the 5′ and 3′ flap endonuc-leases allows DNA strand extension past the lesion. To con-tinue replication through sites of DNA damage, cells useTLS polymerases that are specialized to replicate through aspecific type of DNA lesion (25). The protein proliferatingcell nuclear antigen (PCNA) plays an essential role in thisswitch from replicative to TLS polymerase. PCNA operatesas scaffold to which TLS polymerases bind for DNAunwinding and synthesis (26). The REV1 protein, a TLSpolymerase, is a eukaryotic member of the Y family ofDNA polymerases and functions as a deoxycytidyl transfer-ase, inserting cytidine nucleotides opposite to any templatestrand nucleotide and abasic sites during TLS-mediated rep-lication (27). After translesion synthesis, the replication forkis further stabilized and repaired by homologous recombina-

tion events mediated by proteins such as BRCA1 andBARD1 (BRCA1-associated ring domain protein 1) whichcontains ubiquitin ligase activity and is necessary for properlocalization of RAD51, a central player in HR (28). Directinteraction of BRCA2 with RAD51 promotes its localizationto specific sites of recombination (29). After HR completion,replication can be re-established.

Late S phase/G2 phase

During this phase, FANCM-mediated regression of both thereplication fork occurs followed by HR-mediated fork stabil-ization. When second replication fork has encountered theICL from the opposite direction, unhooking at the 3′ sidecuts the leading strand template. The extension of the lefthand leading strand would lead to its joining with thelagging strand of the right hand fork, followed by secondend capture and extension of the right hand leading strandand left hand template strand. This process would completereplication without the re-initiation of lagging strand synthe-sis, but could generate a double holliday junction that canbe dissolved by BTR complex, comprised of the BLMRECQ helicase, topoisomerase IIIa (TOPOIIIa), RecQ-mediated genome instability I (RMI1), and RecQ-mediatedgenome instability 2 (RMI2) (30) or may in some instancesbe cut by structure-specific nucleases, FAN1. Although HR-mediated repair in S phase promotes error-free DNA, analternative mechanism that is known as non-homologous endjoining (NHEJ) exists to repair broken DNA in all phases of

Figure 1 Schematic diagram of role of Fanconi anemia proteins in interstrand cross-link at various stages of cell cycle.


Shukla et al. DNA interstrand cross-link repair in FA

cell cycle. NHEJ rejoins the two free ends of DNA by bind-ing of the KU70–KU80 heterodimer to the free dsDNA endsand involves the activation of downstream steps by the bind-ing of DNA-dependent protein kinase catalytic subunit(DNA-PKcs) (31). DNA is processed to remove any 5′- or3′ ssDNA tails, and the resulting end is directly rejoinedwith a similarly processed sequence by DNA ligaseIV-XRCC4 (32).Hlavin et al. (33) discussed in their review the initiation

of ICL repair mechanism in mammalian cells in three differ-ent contexts (replication coupled, transcription coupled, andglobal genome ICL repair) and showed that numerous pro-teins are involved in these processes (Table 1). Experimentalstudies using FA cell lines and extracts suggest that FAproteins are not required for the initial steps that process thecross-link but they sense stalled replication forks created byICLs; they help recruit repair proteins to this site and remo-del the stalled replication fork to allow repair to proceed(34, 35). However, certain FA proteins such as FANCJ andFANCM have helicase and nuclease activities; thus, it ispossible that some of these proteins are involved in the ini-tial steps of repair. Mechanistic details of ICL repair bystudies using purified proteins revealed that the activity ofpurified XPF-ERCC1 appears to occur exclusively on sub-strates that mimic stalled replication forks. It has recentlybeen shown that FANCD2 is monoubiquitinated independentof processing by XPF-ERCC1 (36). XPF-ERCC1 was shownto cooperate with proteins in the FA pathway and thescaffolding protein aIISp. Cross-link repair synthesis (CRS)assays in mammalian cell extracts suggested that homolo-gous recombination (HR) was not involved in the repair ofthe ICLs in the extracts, rather repair synthesis may occurby some form of break-induced replication (BIR), whichonly requires limited extents of homology. These assays alsorevealed that XPF-ERCC1 (but not other NER factors) wasrequired for the incisions, and MutSb, and not MutS a orMutL, is involved to specifically recognize psoralen ICLsand create the incisions surrounding the ICL. Overall, mech-anistic studies have demonstrated that the initial recognitionand processing step can be influenced by the level of distor-tion induced by an ICL and repair synthesis step can be

influenced by the chemical structure of an ICL (37, 38).Moreover, ICL repair seems to be a complicated process andinvolves not only different pathways depending on the phaseof the cell cycle, but that even during the G1 phase, multiplepathways may interact to repair ICLs.

Interstrand cross-linking agents and their

therapeutic implications

Many drugs including platinum compounds, nitrogen mus-tards, and aziridines have been successful as chemotherapeuticagents for treating various types of cancers (Table 2).

Platinum compounds

Platinum compounds such as cisplatin, carboplatin, oxalipla-tin, and satraplatin are widely used as chemotherapeuticagents. Cisplatin, the generic name of cis-diamminedichloro-platinum (II), is one of the most frequently used anticancerdrugs and the most effective component used in the combi-nation drug protocols to treat various malignancies such astesticular, ovarian, and lung cancer (39). The major delayedtoxic effects of cisplatin are ototoxicity, nephrotoxicity,peripheral neuropathy (39), and nephrotoxicity often beingdose-limiting toxicity is manifested by decrease in creatinineclearance and electrolyte imbalances (40). Therapy withnephrotoxic drugs such as aminoglycoside antibiotics canaugment the nephrotoxicity induced by cisplatin. Studies inrats and human trials demonstrated that mannitol-induceddiuresis can decrease the nephrotoxicity of cisplatin (41, 42).This is because mannitol is not reabsorbed in the renaltubule and hence increases the osmolality of the glomerularfiltrate, which facilitates excretion of water, and inhibits therenal tubular reabsorption of sodium, chloride, and other sol-utes. Therefore, mannitol promotes the urinary excretion oftoxic materials and protects against nephrotoxicity bypreventing the concentration of toxic substances in the tubu-lar fluid. Thiol-containing compounds such as thiosulfate,diethyldithiocarbamate, and organic thiophosphates havebeen administered in combination with cisplatin to preventnephrotoxicity (40). These compounds reduce the toxicity bydonating a protective thiol group, an effect that is highlyselective for normal but not malignant tissue (43). They limittoxicity by binding to free radicals (44). They may also bindto and detoxify platinum agents by reduction in platinum–DNA adduct formation (45). Cisplatin also inducesmyelosuppression but the degree of leukocytopenia andthrombocytopenia is usually moderate. Carboplatin has thesame antitumor activity as cisplatin but has the advantage ofless toxicity than cisplatin (46). At usual dosage, it is notnephrotoxic, neurotoxic, ototoxic and thus can be adminis-tered to patients who cannot receive cisplatin. Its dose-limit-ing toxicity is myelosuppression, a side effect that is not amajor problem with cisplatin administration. Because car-

Table 1 Proteins involved in interstrand cross-link recognition

ICL repair Proteins

Replication coupled XPF-ERCC1, Mus81-Eme1/Mms4,

hMutSb, RPA, WRN, PCNA, FA proteins,

BRCA1, BRCA2

Transcription coupled XPA, XPB, XPC, XPD, XPF, XPG, CSA, CSB

Global genome NER proteins, FA proteins, aIISp, RPA,

WRN, hMutSb

ICL, Interstrand cross-links; FA, Fanconi anemia; NER, nucleotide

excision repair; PCNA, proliferating cell nuclear antigen.



Table

2Structure,characteristics,andclinicalim

plicationsofDNA

interstrandcross-linkingdrugs

Drugs

Structure

Principle

route

ofadministration

Plasma

half-life

Pharm

acologicalcharacters

Acute

anddelayedtoxicity

Clinicalapplications

Platinum

compounds

Cisplatin

Intravenous,

intraperitoneal

20–4

0min

Bindsextensively

(90%)to

plasmaprotein

andrenaltissue(nephrotoxic),thirty

percent

recoveredin

urinewithin

thefirst24hrs

Nausea,vomiting,anaphylacticreactions,

nephrotoxicity,ototoxicity,peripheral

neuropathy,bonemarrow

depression

Testicular,ovarian,

non-small-cell

lungcancer

Carboplatin

Intravenous

2–3

hLessdrugreacts

withtheplasmaprotein

and

kidneytissueandmore

isexcretedin

the

urine(60–7

0%)

Moderate

nauseaandvomiting,

bonemarrow

depression,low

potential

forototoxicityandneurotoxicity

Ovariancancer

Oxaliplatin

Intravenous

12min

More

reactivethancisplatin,clearancerate

is

2-fold

greaterthanthatofcisplatin

Nausea,vomiting,allergic

reactions,

and/ordiarrhea,neuropathy,ototoxicity,

neutropenia

Colorectalcancer

Satraplatin

Oral

100h

Largely

boundto

bloodcomponents

and

plasmaproteins;littledrugappears

asfree

platinum

intheplasmaultrafiltrate.The

clearancehighly

dependentonrenalfunction.

Nausea,diarrhea,vomiting,bonepain,

anorexia

andarthralgia.Neurotoxicityand

ototoxicitywere

rare

andofmild

or

moderate

severity.

Prostate,lung,

ovarian,and

breast

cancer

Nitrogenmustards

Cyclophosphamide

(CPA)

Intravenous

andoral

3–1

2h

More

than60%

ofmetabolitesare

boundto

plasmaproteins.More

than75%

bioavailable

afteroraladministration.Low

protein

binding,

reversibly

induceitsownmetabolism.Urinary

excretioncomplete

in24h.5–2

5%

is

excretedin

urineasunchangeddrug.

Nausea,vomiting,diarrhea,

darkeningoftheskin/nails,alopecia

(hairloss),amenorrhea,hemorrhagic

cystitis

Chronic

lymphocytic

leukemia

Ifosfamide(IFO)

Intravenous

andoral

60–8

0%

in

72h

Distributionis

more

extensivewithlower

plasmaprotein

bindingcomparedwithCPA.

Renalclearanceis

only

minor.

Alopecia,nausea/vomiting,CNStoxicity,

infection,hemorrhagic

cystitis.

Dose-lim

iting

toxicityis

myelosuppression

Non-small-cell

lung

cancer

Melphalan

Intravenous

andoral

1.5

�0.8

hProtein

bindingis

60–9

0%

(~30%

irreversibly

bound).Cerebrospinalfluid

(CSF)penetration

islow,undergoeshydrolysis

toform

monohydroxyanddihydroxymelphalan.The

24-h

urinary

excretionwas10%

�4.5%,

suggestingthatrenalclearanceis

notamajor

route

ofelim

inationofparentdrug

Nauseaandvomiting,andoralulceration,

severe

allergic

reactions,hairloss,bone

marrow

suppression

Multiple

myeloma,

melanoma,

ovarian

cancer

Chlorambucil

Oral

1.5

hRapidly

andcompletely

absorbedfrom

the

gastrointestinaltract.Extensively

boundto

plasmaandtissueproteins.In

vitro,99%

boundto

plasmaproteins,specifically

albumin.15–6

0%

appears

intheurineafter24h.

Nauseaandvomiting.,drugfever,

hepatotoxicity,infertility,seizures,

gastrointestinaltoxicity,secondary

malignancies,pancytopenia,and

neurotoxicity

Chronic

lymphocytic

leukemia (con

tinued)



Tab

le2.

(con

tinu

ed)

Drugs

Structure

Principle

route

ofadministration

Plasma

half-life

Pharm


Acute

anddelayedtoxicity


Aziridines

Mitomycin

CIntravenous

<1h

Bioreductivealkylation.Could

bedetected

only

duringthe8hrfollowingdrug

administration.Urinary

recovery

waslim

ited

toamaxim

um

of15%

oftheadministered

dose.Renalexcretionis

notamajorroute

of

elim

ination.

Nausea,vomiting,alopecia,stomatitis,

diarrhea,interstitialpneumonitis,

cardiotoxicity,nephrotoxicity,hepatotoxicity,

hemolyticuremic

syndrome,bonemarrow

depression

Esophagealand

bladdercancer

Thiotepa

Intravesical

<1h

Poorlyabsorbedfrom

thegastrointestinal

tract.60–8

5%

recoveredin

urinewithin

24h.

Absorptionfrom

thebladderenoughto

cause

myelosuppression

Anorexia,nausea,vomiting,bonemarrow

depression

Papillary

carcinoma

ofthebladder

Alkanesulfonates

Busulfan

Oral

2.5

hWellabsorbedfrom

thegastrointestinaltract.

Lessthan50%

recoveredin

theurine

Nausea,vomiting,pulm

onary

infiltrationand

fibrosis,diarrhea,im

potence,sterility,

amenorrhea,bonemarrow

depression

Chronic

granulocytic

leukemia

Nitrosoureas

Carm

ustine

Intravenous

15–3

0min

Rapidly

absorbedfrom

thegastrointestinal

tract.Very

lipophilic,rapid

metabolism,easily

penetratesinto

brain

tissue

Nausea,vomiting,hypotension,tachycardia,

pulm

onary

fibrosis,renaldamage,reversible

hepatotoxicity,CNStoxicity,leukemia

Brain

tumor,

Hodgkin’s

disease,

melanoma,

non-Hodgkin’s

lymphomas,

myeloma,lung,and

colorectalcancer

Lomustine

Oral

1–3

h(for

active

metabolites)

Rapidly

absorbedfrom

thegastrointestinal

tract.Very

lipophilic,rapid

metabolism,easily

penetratesinto

brain

tissue,rapidly

hydroxylatedto

products

thatretain

cytotoxic

activity


onary

fibrosis,renal

damage,hepatotoxicity,CNStoxicity,

leukemia,delayedandcumulative

thrombocytopenia,andleucopenia.

Brain

tumor,

Hodgkin’s

disease,

melanoma,

non-Hodgkin’s

lymphomas,

myeloma,lung,and

colorectalcancer

Semustine

Oral

1–3

h(for

active

metabolites)

Rapidly

absorbedfrom

thegastrointestinal

tract.Very

lipophilic,rapid

metabolism,easily

penetratesinto

brain

tissue,rapidly

hydroxylatedto

products

thatretain

cytotoxic

activity


onary

fibrosis,renal

damage,reversible

hepatotoxicity,CNS

toxicity,leukemia,delayedandcumulative

thrombocytopenia,andleucopenia

Brain

tumor,

Hodgkin’s

disease,

melanoma,

non-Hodgkin’s

lymphomas,

myeloma,lung,and

colorectalcancer

Streptozotocin

Intravenous

15min

Bioavailability

is17–2

5%.Distributedand

retainedin

bcells

ofisletofLangerhans

Nauseaandvomiting,renaldamage,

hepatotoxicity,hyperglycemia,hypoglycemia,

anemia

Metastaticcancer

ofthepancreatic

isletcells (con

tinued)



boplatin is not more active as an anticancer drug thancisplatin, and because it induces myelosuppression, its rolein therapy is limited to that of an alternative to cisplatin.Other platinum compounds are satraplatin, which is used totreat prostate and breast cancer (47), and oxaliplatin fortreating colorectal cancer (48). Clinical trials using picoplatinare underway for treatment for lung cancer, solid tumors,prostate, and colorectal cancer (3, 49).

Nitrogen mustards

Cyclophosphamide, ifosfamide, melphalan, and chlorambucilare commonly used nitrogen mustard compounds in chemo-therapy. Cyclophosphamide is the most commonly usedalkylating agent having wider application in cancer chemo-therapy for lymphomas, myelomas, and carcinomas of thebreast, lung, ovary, and endometrium. The dose-limitingtoxicity of cyclophosphamide is bone marrow suppression.As other alkylating agents, this drug is carcinogenic, muta-genic, and teratogenic. The major side effect of cyclophos-phamide is hemorrhagic cystitis (40). Cyclophosphamide isexcreted into the urine as active alkylating agent phosphora-mide mustard, and acrolein and hemorrhagic cystitis appearto be caused by acrolein (40). Ifosfamide, a drug used intreating non-small-cell lung cancer, has antitumor activitysimilar to that of cyclophosphamide. It has dose-limiting tox-icity of bone marrow suppression but as compared to cyclo-phosphamide, it is less myelosuppressive. Ifosfamide cancause Fanconi syndrome like renal damage (50). Neurotoxic-ity has been reported in 30% patients receiving high dosageof ifosfamide. Melphalan, which is principally used to treatmultiple myeloma also, has its clinical implications in treat-ing melanoma, ovarian, and breast cancer (40). The dose-limiting toxicity of melphalan is bone marrow suppression.Chlorambucil is used primarily to treat chronic lymphocyticleukemia (51) and occasionally to treat lymphomas andbreast (52) or ovarian carcinomas (53).

Aziridines

These compounds contain three membered aziridine ringsand found to be active as anticancer drugs. Mitomycin C, anantibiotic derived from streptomyces species, acts through aunique mechanism as a bifunctional alkylator. It is activatedby the chemical or enzymatic reduction of its quinone group.It has been used to treat cancers of stomach, colon, pancreas,breast, lung, head, and neck (40). Remissions after therapyare generally limited and of short duration. Mitomycin, thegeneric name of mitomycin C, is mutagenic and carcino-genic and also produces chromosome breakage. Mitomycinforms ICLs with DNA after reduction of quinone moiety,which breaks C-1 aziridine ring creating a semiquinoneradical at C-10 that reacts with nucleophilic groups of DNA(most commonly guanine) (40). Due to the formation ofT

able

2.(con

tinu

ed)

Drugs

Structure

Principle

route

ofadministration

Plasma

half-life

Pharm


Acute

anddelayedtoxicity


Methylatingagents

Dacarbazine

Intravenous

40min

Absorptionis

slow

andincomplete.Little

plasmabinding(20%),40%

isexcretedin

urineunchangedin

6h

Nausea,vomiting,diarrhea,anaphylaxis,

impairedrenalfunction,flu-likesyndrome,

hepaticnecrosis,photosensitivity,bone

marrow

depression

Metastatic

malignant

melanoma,

Hodgkin’s

disease,

softtissuesarcomas

Procarbazine

Oral

7min

Rapidly

andcompletely

absorbedfrom

the

gastrointestinaltract.Metabolizedby

cytochromeP-450,70%

recoveredin

urinein

24hin

theform

ofmetabolite.<5%

excrete

asparentdrug

Mild

nausea,vomiting,CNSdepression,

peripheralneuropathy,pulm

onary

infiltration,

azoosperm

ia,leukemia,bonemarrow

depression

AdvancedHodgkin’s

diseases,

non-Hodgkin’s

lymphomas,

small-celllung

carcinomas,

malignantmelanoma



ICLs, a protective effect could take place by a mechanismsuperimposed on the repair process. Resistance for mitomy-cin can occur due to decreased reductive activation andincreased excision repair. Tumor cells resistant to mitomycinhave shown decreased drug uptake, cross-resistance withalkaloids and anthracyclines, and reversion of resistance withverapamil confirming multidrug resistance (MDR) pheno-type.High doses of mitomycin cause interstitial pneumonitis,

nephrotoxicity, and cardiotoxicity. Nephrotoxicity may berelated to immune complex formation, while interstitialpneumonitis and cardiotoxicity may be due to reactive oxy-gen species as a result of auto-oxidation of the reduced qui-none under aerobic conditions (40). Leukopenia andthrombocytopenia are the major dose-limiting toxicity bymitomycin C. Thiotepa, another drug in this group, is nowonly rarely administered systemically as it produces bonemarrow depression as a major toxic effect. The drug hasfound its clinical implication in treating localized bladdercarcinoma (54).

Alkane sulfonates

Alkane sulfonates such as busulfan, a bifunctional alkylatingagent, is used in treating chronic granulocytic leukemia (55).It is also been used in high-dose combination with cyclo-phosphamide in conditioning regimens for allogenic bonemarrow transplantation (56). Resistance to busulfan appearsto reflect more rapid removal of the DNA cross-links. Themost common toxic effects of busulfan, bone marrowdepression, or pulmonary toxicity are dose-limiting (40).

Nitrosoureas

A large number of nitrosourea compounds have shown theirapplications as antitumor agents.Carmustine, lomustine, and semustine are nitrosoureas;

they are lipophilic in nature and distribute widely in tissues.Their lipophilic nature permits them to cross the blood–brainbarrier into the cerebrospinal fluid (CSF). Thus, they havethe important clinical application in treating brain tumorsand meningeal leukemia. They are also used in treatingHodgkin’s disease and melanoma and secondary therapy ofnon-Hodgkin’s lymphomas, myeloma, lung, and colorectalcancers (40). Streptozotocin, another drug in this group, hasits important aspects of its retention in ß cells of the Langer-hans islets and is thus employed clinically in the treatmentfor metastatic islet cell carcinoma of the pancreas (57).Studies demonstrated that streptozotocin destroys insulin-producing B cells and thus is diabetogenic (58). The diabe-togenic action of streptozotocin in animals is mediatedthrough a reduction of nicotinamide adenine dinucleotide(NAD) in pancreatic cells (59). Streptozotocin causes DNAdamage through its alkylation activity, which is repaired by

an excision repair process, and requires the activation of theNAD-dependent enzyme poly (ADP-ribose) synthetase (60).This enzyme is continuously activated in the beta cell, andhence, streptozotocin leads to the depletion of NAD. Thecontinuous loss of NAD leads to a cessation of cellular func-tion and eventually cell death (60). In addition to streptozo-tocin-induced cytotoxicity through DNA alkylation, reactiveoxygen species (O2•-, H2O2, HO•, NO•) play a critical rolein the mechanism of DNA damage and cytotoxicity of strep-tozotocin (61). The major toxic effect of nitrosoureas is leu-copenia and thrombocytopenia. Bone marrow toxicity isdelayed, dose-limiting and cumulative. Nitrosoureas areoccasionally nephrotoxic and hepatotoxic and are carcino-genic and mutagenic in nature (40, 62).

Methylating agents

Methylating agents such as dacarbazine are used in singledrug and combination drug treatment for metastatic malig-nant melanoma (63). In combination with other drugs,dacarbazine is also used in the treatment for Hodgkin’s dis-ease (64). The most common delayed toxicity in case of da-carbazine is bone marrow depression, which is dose-limiting. Procarbazine, a methylating drug, has been used incombination drug therapy to treat advanced Hodgkin’s dis-ease (65). Because procarbazine has ability to enter the CSF,it can also find its application in treating malignant braintumors (66). The major toxicity of procarbazine is bone mar-row depression with leucopenia and thrombocytopenia.These agents produce methylation of DNA, predominantlyon the O-6 and N-7 positions of guanylic acid. The lesionsproduce both spontaneous and enzyme-mediated single-strand breaks (67, 68) that are cytotoxic. However, it hasnow been demonstrated that active mismatch DNA repair isa major mediator of the cytotoxicity of these agents.

Cross-linker toxicity and redox

biotransformation in FA

Several studies on cross-linking toxicity of FA-related xeno-biotics such as MMC, cisplatin, melphalan, 8-methoxypsora-len+UVA, and cyclophosphamide (CP) have been reportedto be associated with redox-related mechanisms includingbiotransformation (69–74). Joenje and Oostra in 1986 (75)found that FA cells sensitive to CP and it is suggested thatthe FA cells were endowed with the activities effecting CPbiotransformation to its active metabolites. The cytochromeP450 (CYP) enzyme family also related to redoxmechanisms as CYP activities involved with xenobioticdetoxification and generating mutagenic metabolites (76).The FANCC protein was found to be associated with redox-related activities, namely NADPH cytochrome P450 reduc-tase (77) and GST (78). The FANCG protein interacts witha P450 protein, cytochrome P450 2E1 (CYP2E1), an activity



also known to be involved in redox biotransformation of xe-nobiotics including MMC (79). The DEB is much commonalkylating agent and potent DNA cross-linker. The DEBexhibits a different toxicity mechanism as it has the peculiar1,3-diepoxidation pattern in which the ring tension accountsfor the marked reactivity toward nucleophiles and the doubleepoxide ring allows for the formation of ICLs with manyDNA sequences (80). However, the genotoxic and cytotoxiceffects of DEB depend on oxygen levels and result in deple-tion of dose-related reduced glutathione (GSH) (81); thus,two glutathione-related activities such as glutathione-s-trans-ferase (GST) and GSH peroxidase (GPx) have role in thedetoxification of DEB-induced DNA damage (82). It wasalso been demonstrated that both DEB and MMC exposuresinduce concentration-related DNA oxidative damage[8-hydroxy-deoxyguanosine (8-OHdG)] (83). Reuter andcolleagues (84) identified 69 proteins as direct interactors ofFANCA, FANCC, or FANCG that were associated withtranscription regulation, signaling, oxidative metabolism, andintracellular transport. The studies on double knockout miceof Fanca-/-, Trp53-/- reported that deficiency in the Fancagene in mice elicits a p53-dependent growth arrest and DNAdamage response to oxidative DNA; oxidative stress inducesp53 response in Fanca-/-cells, likely due to accumulation ofunrepaired DNA damage (85). Some studies suggested thatthe activation of p53 leads to an increase in reactive oxygenspecies (ROS) that, by possibly interfering with mitochon-drial function and integrity, leads to cell death (86).Although few studies reported mitochondrial dysfunctionand oxidative stress, there is always a scope to study thesame at molecular level.

Modulation of ICL sensitivity for therapeutic

interventions

The ability to change cell sensitivity to cross-linking drugscould have enormous therapeutic implications. However, theclinical implications of ICL-based chemotherapy arerestricted by dose-limiting toxicities in the blood. Under-standing the mechanism of how tumor cells become resistantto ICLs and in applying this information to promote resis-tance in the non-diseased cells of the blood may help in pre-vention of toxicity. Methods targeting increase in ICL repairin normal tissue might lead to effective chemoprotection.Conversely, targeting inactivation of ICL repair by methodsthat lead to increase resistance to ICL-inducing drugs willhelp in controlling tumors, for example, cisplatin resistancewas observed in ovarian tumors after FANCF demethylationand re-expression (11). Inhibition of extracellular signalingpathways can inactivate ICL repair in tumor cells. For exam-ple, therapy with trastuzumab leads to 35–40% reduction inrepair of cisplatin–DNA adducts and promotes drug-inducedkilling in target cells (87) by abrogating the PI3K/Akt sig-naling pathway (88).

DNA repair–deficient tumor cells have been shown toaccumulate high levels of DNA damage. Therefore, thesecells become hyperdependent on DNA damage responsepathways, including the CHK1-kinase-mediated response,suggesting that DNA repair–deficient tumors should exhibitincreased sensitivity to CHK1 inhibition (89). CHK1functions by phosphorylating several key cell cycle proteins,thereby halting the transition of cells from G2 to M phase.Thus, the absence of CHK1 allows tumor cells to enter mito-sis even though they have extensive DNA damage. Thera-peutic strategies aiming at CHK1 inhibition or MAPKinhibition (MAPK participates in a parallel pathway toCHK1 in G2) are shown to be promising in re-establishingcisplatin sensitivity in tumor cells (90). Several CHK1 inhib-itors are currently undergoing clinical trials as antineoplasticagents (91, 92). These inhibitors are used largely in combi-nation with other DNA-damaging agents including cisplatin(93) fluorouracil (94), topotecan (95), and cytarabine (96).So far, the FA pathway has received most of the attention asa target due to the specific sensitivity of FA-deficient cellsto cross-linking agents. Disruption of FA core complexproteins FANCC and FANCG in adenocarcinoma cell linesabrogates FANCD2 monoubiquitylation and renders cellssensitive to agents such oxaliplatin and melphalan (97, 98).Screening for inhibitors of the FA pathway identified smallmolecules such as wortmannin, H-9, and alsterpaullone andthe natural product curcumin that are selectively toxic toFA-deficient cells (97–100). These FA inhibitors can be usedboth as a chemosensitizers in cisplatin-based cancer treat-ment and as synthetic lethal agents in tumors with defects inthe FA genes. Moreover, if any other pathway is hyperde-pendent on FA pathway, inhibitors of that particular pathwaymay activate FA/BRCA pathway and may act as therapy fortumors deficient in FA/BRCA pathway, for example, inhibi-tors of PARP-1 that are involved in base excision repair, arepair pathway hyperdependent on FA/BRCA pathway. Clin-ical trial with a PARP-1 inhibitor, olaparib, has shownPARP-1 inhibition as useful for the treatment of patientswith BRCA2-deficient ovarian cancer (101). PARP-1 inhibi-tors alone or in combination with cisplatin have also shownpromising results against BRCA-deficient cancers (102). Fur-thermore, improved responsiveness to platinum and PARP-1inhibitors has also been observed in a subset of patients withsporadic epithelial ovarian cancers (103). Similarly, PARP-1inhibitors may be a successful therapeutic strategy for asubset of patients with AML deficient in HR such as FA-deficient pathway or deficient in RAD51 loci (104).Because the FA/BRCA pathway counteracts the DNA inter-

strand cross-link genotoxicity related to endogenous alde-hydes, bone marrow failure in patients with FA might bemitigated if detoxification of aldehydes could be enhanced(13, 105). The only curative option to date for the hematopoi-etic signs in patients with Fanconi anemia is bone marrowtransplant. Regarding gene therapy, if HSPCs would be col-



lected at an early age to avoid accumulation of DNA damage,it can be a promising therapeutic option for patients with FA(106).

Future perspectives

The repair of ICLs is mediated by assembly of many com-ponents from different repair pathways. Understanding thenetwork of interactions between signaling pathways andrepair pathways triggered by ICLs together and their mecha-nisms of action has yet to be completely elucidated. Thestudy of ICL repair using development of new methods tosynthesize oligonucleotides containing site-specific ICLs willhelp understanding the molecular basis of how ICL repairdiffers from repair pathways of lesions that only affect onestrand of DNA. These insights should provide opportunitiesto find new targets for drug developments to increase thetherapeutic efficiency of cross-linking agents and to targettumor cells with specific defects in ICL repair. Better under-standing of the mechanism of action of FA pathway inhibi-tors, emerging structural studies of FA proteins, and themechanisms by which FA proteins contribute to ICL repairwill provide additional opportunities for inhibitors that targetFA proteins. As discussed above, reducing DNA damage inHSCs caused by endogenous reactive metabolites (alde-hydes) has implications to develop future therapeutic inter-ventions and clinical management for patients with FA.

Conflict of interest

There is no conflict of interest involved with this article.

References

1. Vasquez KM, Legerski RJ. DNA interstrand crosslinks:repair, cell signaling, and therapeutic implications. EnvironMol Mutagen 2010;51:491–2.

2. Guainazzi A, Scharer OD. Using synthetic DNA interstrandcrosslinks to elucidate repair pathways and identify newtherapeutic targets for cancer chemotherapy. Cell Mol LifeSci 2010;67:3683–97.

3. Deans AJ, West SC. DNA interstrand crosslink repair andcancer. Nat Rev Cancer 2011;11:46780.

4. Auerbach AD, Buchwald M, Joenje H. Fanconi anemia, In:Scriver CR, Sly WS, Childs B, et al., eds. The Metabolicand Molecular Bases of Inherited Disease. Vol 1, 8th edn.New York: McGraw-Hill; 2001:753–68.

5. D’Andrea AD, Grompe M. Molecular biology of Fanconianemia: implications for diagnosis and therapy. Blood1997;90:1725–36.

6. Svahn J, Dufour C. Fanconi anemia -learning from children.Pediatr Rep 2011;3:e8.

7. Tulpule A, Lensch MW, Miller JD, et al. Knockdown ofFanconi anemia genes in human embryonic stem cells

reveals early developmental defects in the hematopoieticlineage. Blood 2010;115:3453–62.

8. Dhillon VS, Shahid M, Husain SA. CpG methylation of theFHIT, FANCF, cyclin-D2, BRCA2 and RUNX3 genes inGranulosa cell tumors (GCTs) of ovarian origin. Mol Cancer2004;3:33.

9. Marsit CJ, Liu M, Nelson HH, et al. Inactivation of the Fanconianemia/BRCA pathway in lung and oral cancers: implicationsfor treatment and survival. Oncogene 2004;23:1000–4.

10. Narayan G, Arias-Pulido H, Nandula SV, et al. Promoterhypermethylation of FANCF: disruption of Fanconi Ane-mia-BRCA pathway in cervical cancer. Cancer Res2004;64:2994–7.

11. Taniguchi T, Tischkowitz M, Ameziane N, et al. Disruptionof the Fanconi anemia-BRCA pathway in cisplatin-sensitiveovarian tumors. Nat Med 2003;9:568–74.

12. van der Heijden MS, Yeo CJ, Hruban RH, Kern SE. Fan-coni anemia gene mutations in young-onset pancreatic can-cer. Cancer Res 2003;63:2585–8.

13. O’Brien PJ, Siraki AG, Shangari N. Aldehyde sources,metabolism, molecular toxicity mechanisms and possibleeffects on human health. Crit Rev Toxicol 2005;35:609–62.

14. Nagayoshi H, Mastumoto A, Nishi R, Kawamoto T, IchibaM, Matsuda T. Increased formation of gastric N2-ethylid-ene-29-deoxyguanosine DNA adducts in aldehyde dehydro-genase-2 knockout mice treated with ethanol. Mutat Res2009;673:74–7.

15. Matsuda T, Matsumoto A, Uchida M, et al. Increased for-mation of hepatic N2-ethylidene-29-deoxyguanosine DNAadducts in aldehyde dehydrogenase-2 knockout mice treatedwith ethanol. Carcinogenesis 2007;28:2363–6.

16. Seitz HK, Stickel F. Molecular mechanisms of alcohol-med-iated carcinogenesis. Nat Rev Cancer 2007;7:599–612.

17. Langevin F, Crossan GP, Rosado IV, et al. Fancd2 counter-acts the toxic effects of naturally produced aldehydes inmice. Nature 2011;475:53–8.

18. Garaycoechea JI, Crossan GP, Langevin F, et al. Genotoxicconsequences of endogenous aldehydes on mouse haemato-poietic stem cell function. Nature 2012;489:571–5.

19. Ceccaldi R, Parmar K, Mouly E, et al. Bone marrow failurein Fanconi anemia is triggered by an exacerbated p53/p21DNA damage response that impairs hematopoietic stem andprogenitor cells. Cell Stem Cell 2012;11:36–49.

20. Svendsen JM, Smogorzewska A, Sowa ME, et al. Mamma-lian BTBD12/SLX4 assembles a Holliday junction resolvaseand is required for DNA repair. Cell 2009;138:63–77.

21. Green AM, Kupfer GM. Fanconi anemia. Hematol OncolClin North Am 2009;23:193–214.

22. Meetei AR, Medhurst AL, Ling C, et al. A human orthologof archaeal DNA repair protein Hef is defective in Fanconianemia complementation group M. Nat Genet 2005;37:958–63.

23. Meetei AR, de Winter JP, Medhurst AL, et al. A novelubiquitin ligase is deficient in Fanconi anemia. Nat Genet2003;35:165–70.



24. Smogorzewska A, Desetty R, Saito TT, et al. A geneticscreen identifies FAN1, a Fanconi anemia-associated nucle-ase necessary for DNA interstrand crosslink repair. Mol Cell2010;39:36–47.

25. McCulloch SD, Kunkel TA. The fidelity of DNA synthesisby eukaryotic replicative and translesion synthesis polyme-rases. Cell Res 2008;18:148–61.

26. Andersen PL, Xu F, Xiao W. Eukaryotic DNA damage tol-erance and translesion synthesis through covalent modifica-tions of PCNA. Cell Res 2008;18:162–73.

27. Prakash S, Johnson RE, Prakash L. Eukaryotic translesionsynthesis DNA polymerases: specificity of structure andfunction. Annu Rev Biochem 2005;74:317–53.

28. Polanowska J, Martin JS, Garcia-Muse T, Petalcorin MI,Boulton SJ. A conserved pathway to activate BRCA1-dependent ubiquitylation at DNA damage sites. EMBOJ 2006;25:2178–88.

29. Thorslund T, McIlwraith MJ, Compton SA, Lekomtsev S,Petronczki M, Griffith JD, West SC. The breast cancertumor suppressor BRCA2 promotes the specific targeting ofRAD51 to single-stranded DNA. Nat Struct Mol Biol2010;17:1263–5.

30. Wu L, Hickson ID. The Bloom’s syndrome helicasesuppresses crossing over during homologous recombination.Nature 2003;426:870–4.

31. Gottlieb TM, Jackson SP. The DNA-dependent proteinkinase: requirement for DNA ends and association with Kuantigen. Cell 1993;72:131–42.

32. Lieber MR, Ma Y, Pannicke U, Schwarz K. Mechanism andregulation of human non-homologous DNA end-joining.Nat Rev Mol Cell Biol 2003;4:712–20.

33. Hlavin EM, Smeaton MB, Miller PS. Initiation of DNA in-terstrand cross-link repair in mammalian cells. Environ MolMutagen 2010;51:604–24.

34. Niedernhofer LJ, Lalai AS, Hoeijmakers JH. Fanconi ane-mia (cross) linked to DNA repair. Cell 2005;123:1191–8.

35. Thompson LH, Hinz JM. Cellular and molecular conse-quences of defective Fanconi anemia proteins in replication-coupled DNA repair: mechanistic insights. Mutat Res2009;668:54–72.

36. Bhagwat N, Olsen AL, Wang AT, et al. XPF-ERCC1 par-ticipates in the Fanconi anemia pathway of crosslink repair.Mol Cell Biol 2009;29:6427–37.

37. Smeaton MB, Hlavin EM, McGregor Mason T, et al.Distortion-dependent unhooking of interstrand cross-linksin mammalian cell extracts. Biochemistry2008;47:9920–30.

38. Smeaton MB, Hlavin EM, Noronha AM, Murphy SP, WildsCJ, Miller PS. Effect of cross-link structure on DNA inter-strand cross-link repair synthesis. Chem Res Toxicol2009;22:1285–97.

39. Giaccone G. Clinical perspectives on platinum resistance.Drugs 2000;59:9–17; discussion 37-8.

40. Pratt WB, Ruddon RW, Ensminger WD, Maybaum J. TheAnticancer Drugs. New York: Oxford University Press,1994.

41. Miller RP, Tadagavadi RK, Ramesh G, Reeves WB. Mecha-nisms of Cisplatin nephrotoxicity. Toxins (Basel)2010;2:2490–518.

42. Ries F, Klastersky J. Nephrotoxicity induced by cancer che-motherapy with special emphasis on cisplatin toxicity. AmJ Kidney Dis 1986;8:368–79.

43. Yao X, Panichpisal K, Kurtzman N, Nugent K. Cisplatinnephrotoxicity: a review. Am J Med Sci 2007;334:115–24.

44. Myers CE, McGuire WP, Liss RH, et al. Adriamycin: therole of lipid peroxidation in cardiac toxicity and tumorresponse. Science 1977;197:165–7.

45. Treskes M, van der Vijgh WJ. WR2721 as a modulator ofcisplatin- and carboplatin-induced side effects in comparisonwith other chemoprotective agents: a molecular approach.Cancer Chemother Pharmacol 1993;33:93–106.

46. Smith IE, Harland SJ, Robinson BA, Evans BD, GoodhartLC, Calvert AH, Yarnold J, Glees JP, Baker J, Ford HT.Carboplatin: a very active new cisplatin analog in the treat-ment of small cell lung cancer. Cancer Treat Rep1985;69:43–6.

47. Doshi G, Sonpavde G, Sternberg CN. Clinical and pharma-cokinetic evaluation of satraplatin. Expert Opin Drug MetabToxicol 2012;8:103–11.

48. Yaman E, Uner A, Er O, et al. Capecitabine plus oxalipl-atin (xelox) in the treatment of chemotherapy-naivepatients with metastatic colorectal cancer. Med Oncol2007;24:431–5.

49. Eckardt JR, Bentsion DL, Lipatov ON, Polyakov IS, Mack-intosh FR, Karlin DA, Baker GS, Breitz HB. Phase II studyof picoplatin as second-line therapy for patients with small-cell lung cancer. J Clin Oncol 2009;27:2046–51.

50. Zamlauski-Tucker MJ, Morris ME, Springate JE. Ifosfamidemetabolite chloroacetaldehyde causes Fanconi syndrome inthe perfused rat kidney. Toxicol Appl Pharmacol1994;129:170–5.

51. Catovsky D, Else M, Richards S. Chlorambucil–still notbad: a reappraisal. Clin Lymphoma Myeloma Leuk 2011;11:S2–6.

52. Evers B, Schut E, van der Burg E, et al. A high-throughputpharmaceutical screen identifies compounds with specifictoxicity against BRCA2-deficient tumors. Clin Cancer Res2010;16:99–108. Epub 2009 Dec 15.

53. McGuire WP 3rd, Markman M. Primary ovarian cancer che-motherapy: current standards of care. Br J Cancer 2003;89:S3–8.

54. Agnelli G, de Cunto M, Gresele P, del Favero A. Earlyonset life-threatening myelosuppression after low dose ofintravesical thiotepa. Postgrad Med J 1982;58:380–1.

55. Binet JL. Treatment of chronic lymphocytic leukaemia.French Co-operative Group on CLL. Baillieres Clin Haema-tol 1993;6:867–78.

56. Dreger P, Brand R, Hansz J, et al. Treatment-related mor-tality and graft-versus-leukemia activity after allogeneicstem cell transplantation for chronic lymphocytic leukemiausing intensity-reduced conditioning. Leukemia2003;17:841–8.



57. Kouvaraki MA, Ajani JA, Hoff P, et al. Fluorouracil, doxo-rubicin, and streptozocin in the treatment of patients withlocally advanced and metastatic pancreatic endocrine carci-nomas. J Clin Oncol 2004;22:4762–71.

58. Das A, Mostofa M, Hoque M, et al. Comparative efficacyof neem (azadirachta indica) and metformin hydrochloride(comet� in streptozotocin induced diabetes mellitus in rats.Bang J Vet Med 2010;8:75–80.

59. Weiss RB. Streptozocin: a review of its pharmacology,efficacy, and toxicity. Cancer Treat Rep 1982;3:427–35.

60. Wilson GL, Leiter EH. Streptozotocin interactions with pan-creatic b cells and the induction of insulin-dependent diabe-tes. Curr Topics Micro Immuno 1990;156:27–54.

61. Bolzan AD, Bianchi MS. Genotoxicity of Streptozotocin.Mutat Res 2002;512:121–34.

62. Sheweita SA. Drug-metabolizing enzymes: mechanisms andfunctions. Curr Drug Metab 2000;1:107–32.

63. Spiro T, Liu L, Gerson S. New cytotoxic agents for thetreatment of metastatic malignant melanoma: temozolomideand related alkylating agents in combination with guanineanalogues to abrogate drug resistance. Forum (Genova)2000;10:274–85.

64. Case DC Jr, Young CW, Lee BJ 3rd. Combination chemo-therapy of MOPP-resistant Hodgkin’s disease with adriamy-cin, bleomycin, dacarbazine and vinblastine (ABDV).Cancer 1977;39:1382–6.

65. Bonadonna G, Valagussa P, Santoro A, Viviani S, BonfanteV, Banfi A. Hodgkin’s disease: the Milan Cancer Instituteexperience with MOPP and ABVD. Recent Results CancerRes 1989;117:169–74.

66. Levin VA, Silver P, Hannigan J, Wara WM, Gutin PH,Davis RL, Wilson CB. Superiority of postradiotherapy adju-vant chemotherapy with CCNU, procarbazine, and vincris-tine (PCV) over BCNU for anaplastic gliomas: NCOG6G61 final report. Int J Radiat Oncol Biol Phys1990;18:321–4.

67. Verly WG. Monofunctional alkylating agents and apurinicsites in DNA. Biochem Pharmacol 1974;23:3.

68. Verly WG, Paquette Y. An endonuclease for depurinatedDNA in Escherichia coli B. Can J Biochem1972;50:217–24.

69. Szybalski W, Iyer VN. Crosslinking of DNA by enzymati-cally or chemically activated mitomycins and porfiromycins,bifunctionally “alkylating” antibiotics. Fed Proc1964;23:946–57.

70. Pritsos CA, Sartorelli AC. Generation of reactive oxygenradicals through bioactivation of mitomycin antibiotics.Cancer Res 1986;46:3528–32.

71. Durse L, Rajagopalan S, Eliot HM, Covey JM, et al. DNAinterstrand cross-link and free radical formation in a humanmultidrug resistant cell line from mitomycin C and its ana-logues. Cancer Res 1990;50:648–52.

72. Clarke AA, Philpott NJ, Gordon-Smith EC, et al. The sensi-tivity of Fanconi anaemia group C cells to apoptosisinduced by mitomycin C is due to oxygen radical genera-tion, not DNA crosslinking. Br J Haematol 1997;96:240–7.

73. Penketh PG, Hodnick WF, Belcourt MF, et al. Inhibition ofDNA crosslinking by mitomycin C by peroxidase-mediatedoxidation of mitomycin C hydroquinone. J Biol Chem2001;276:34445–52.

74. Pagano G, Talamanca AA, Castello G, et al. Oxidative stressin Fanconi anemia: from cells and molecules towards pros-pects in clinical managements. Biol Chem 2012;393:11–21.

75. Joenje H, Oostra AB. Clastogenicity of cyclophosphamidein Fanconi’s anemia lymphocytes without exogenous meta-bolic activation. Cancer Genet Cytogenet 1986;22:339–45.

76. Barouki R, Morel Y. Repression of cytochrome P450 1A1gene expression by oxidative stress: mechanisms and bio-logical implications. Biochem Pharmacol 2001;61:511–6.

77. Kruyt FA, Hoshino T, Liu JM, et al. Abnormal microsomaldetoxification implicated in Fanconi anemia group C byinteraction of the FAC protein with NADPH cytochromeP450 reductase. Blood 1998;92:3050–6.

78. Cumming RC, Lightfoot J, Beard K, et al. Fanconi anemiagroup C protein prevents apoptosis in hematopoietic cellsthrough redox regulation of GSTP1. Nat Med2001;7:814–20.

79. Futaki M, Igarashi T, Watanabe S. The FANCG Fanconianemia protein interacts with CYP2E1: possible role inprotection against oxidative DNA damage. Carcinogenesis2002;23:67–72.

80. Vlachodimitropoulos D, Norppa H, Autio K, et al. GSTT1-dependent induction of centromere negative and -positivemicronuclei by 1,2:3,4-diepoxybutane in cultured humanlymphocytes. Mutagenesis 1997;12:397–403.

81. Boogaard PJ, Bond JA. The role of hydrolysis in the detoxi-fication of 1,2:3,4-diepoxybutane by human, rat, and mouseliver and lung in vitro. Toxicol Appl Pharmacol1996;141:617–27.

82. Span�o M, Cordelli E, Leter G, et al. Diepoxybutane cyto-toxicity on mouse germ cells is enhanced by in vivo gluta-thione depletion: a fl ow cytometric approach. Mutat Res1998;397:37–43.

83. Pagano G, Degan P, De Biase A. Diepoxybutane and mito-mycin C toxicity is associated with the induction of oxida-tive DNA damage in sea urchin embryos. Hum Exp Toxicol2001;20:651–5.

84. Reuter TY, Medhurst AL, Waisfisz Q, et al. Yeast two-hybrid screens imply involvement of Fanconi anemia pro-teins in transcription regulation, cell signaling, oxidativemetabolism, and cellular transport. Exp Cell Res2003;289:211–21.

85. Rani R, Li J, Pang Q. Differential p53 engagement inresponse to oxidative and oncogenic stresses in Fanconianemia mice. Cancer Res 2008;68:9693–702.

86. Du W, Adam Z, Rani R, Zhang X, Pang Q. Oxidative stressin Fanconi anemia hematopoiesis and disease progression.Antioxid Redox Signal 2008;10:1909–21.

87. Pietras RJ, Fendly BM, Chazin VR, Pegram MD, HowellSB, Slamon DJ. Antibody to HER-2/neu receptor blocksDNA repair after cisplatin in human breast and ovarian can-cer cells. Oncogene 1994;9:1829–38.



88. Wong AL, Lee SC. Mechanisms of resistance to trast-uzumab and novel therapeutic strategies in HER2-positivebreast cancer. Int J Breast Cancer 2012;2012:415170.

89. Chen CC, Kennedy RD, Sidi S, et al. CHK1 inhibition as astrategy for targeting Fanconi Anemia (FA) DNA repairpathway deficient tumors. Mol Cancer 2009;8:24.

90. Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB. p53-defi-cient cells rely on ATM- and ATR-mediated checkpointsignaling through the p38MAPK/MK2 pathway for survivalafter DNA damage. Cancer Cell 2007;11:175–89.

91. Tse AN, Carvajal R, Schwartz GK. Targeting checkpointkinase 1 in cancer therapeutics. Clin Cancer Res2007;13:1955–60.

92. Zhou BB, Bartek J. Targeting the checkpoint kinases:chemosensitization versus chemoprotection. Nat Rev Cancer2004;4:216–25.

93. Lara PN Jr, Mack PC, Synold T, et al. The cyclin-depen-dent kinase inhibitor UCN-01 plus cisplatin in advancedsolid tumors: a California cancer consortium phase I phar-macokinetic and molecular correlative trial. Clin Cancer Res2005;11:4444–50.

94. Kortmansky J, Shah MA, Kaubisch A, et al. Phase I trial ofthe cyclin-dependent kinase inhibitor and protein kinase Cinhibitor 7-hydroxystaurosporine in combination with Fluo-rouracil in patients with advanced solid tumors. J ClinOncol 2005;23:1875–84.

95. Hotte SJ, Oza A, Winquist EW, et al. Phase I trial ofUCN-01 in combination with topotecan in patients withadvanced solid cancers: a Princess Margaret Hospital PhaseII Consortium study. Ann Oncol 2006;17:334–40.

96. Sampath D, Cortes J, Estrov Z, et al. Pharmacodynamics ofcytarabine alone and in combination with 7-hydroxystaurosp-orine (UCN-01) in AML blasts in vitro and during a clinicaltrial. Blood 2006;107:2517–24.

97. Gallmeier E, Calhoun ES, Rago C, et al. Targeted disrup-tion of FANCC and FANCG in human cancer provides a

preclinical model for specific therapeutic options. Gastroen-terology 2006;130:2145–54.

98. Gallmeier E, Kern SE. Targeting Fanconi anemia/BRCA2 pathway defects in cancer: the significance ofpreclinical pharmacogenomic models. Clin Cancer Res2007;13:4–10.

99. Chirnomas D, Taniguchi T, de la Vega M, et al. Chemosen-sitization to cisplatin by inhibitors of the Fanconi anemia/BRCA pathway. Mol Cancer Ther 2006;5:952–61.

100. Landais I, Sobeck A, Stone S. A novel cell-free screenidentifies a potent inhibitor of the Fanconi anemia pathway.Int J Cancer 2009;124:783–92.

101. Audeh MW, Carmichael J, Penson RT, et al. Oralpoly(ADP-ribose) polymerase inhibitor olaparib inpatients with BRCA1 or BRCA2 mutations and recur-rent ovarian cancer: a proof-of-concept trial. Lancet2010;376:245–51.

102. Evers B, Helleday T, Jonkers J. Targeting homologousrecombination repair defects in cancer. Trends PharmacolSci 2010;31:372–80.

103. Konstantinopoulos PA, Spentzos D, Karlan BY, TaniguchiT, Fountzilas E, Francoeur N, Levine DA, Cannistra SA.Gene expression profile of BRCAness that correlates withresponsiveness to chemotherapy and with outcome inpatients with epithelial ovarian cancer. J Clin Oncol2010;28:3555–61.

104. D’Andrea AD. Targeting DNA repair pathways in AML.Best Pract Res Clin Haematol 2010;23:469–73.

105. Joenje H. Metabolism: alcohol, DNA and disease. Nature2011;475:45–6.

106. Tolar J, Adair JE, Antoniou M, et al. Stem cell genetherapy for Fanconi anemia: report from the 1st interna-tional Fanconi anemia gene therapy working group meeting.Mol Ther 2011;19:1193–8.



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DNA interstrand cross-link repair: understanding role of Fanconi anemia pathway and therapeutic implications