Review of the clinical experience with 5-azacytidine and 5-aza-2'-
`deoxycytidine in solid tumors
`Ana Aparicio* & Jeffrey S Weber
`Address
`Division of Medical Oncology
`University of Southern California
`Norris Comprehensive Cancer Center
`1441 Eastlake Avenue
`Los Angeles
`CA 90033
`USA
`Email: aaparici@usc.edu
`
`627
`
`mature oocytes and sperm are wiped out with a genome-
`wide wave of demethylation. Then, selective de novo
`methylation takes place, sparing the housekeeping genes
`and those that need to be active during embryogenesis.
`Finally,
`tissue-specific
`genes
`are
`demethylated in
`of
`association with the onset
`their
`activity. which
`presumably
`leads
`to
`tissue
`differentiation. DNA
`methylation is also involved in the inactivation of the X
`chromosome in females and in parental imprinting, and it
`probablycontributes to the aging process [5,6].
`
`Changes in methylation are among the most common
`genomic alterations found in neoplasia. On one hand, there
`is global hypomethylation
`of
`the DNA leading
`to
`chromosomal
`instability and an increased rate of genetic
`mutations [7e]. On the other, there is hypermethylation of
`CpGislands located in the promoters of tumor suppressor
`genes such as p16, p13. VHL and Rb. that renders them silent
`
`and provides a growth advantage for the cell
`[8]. In this
`regard,
`it
`is interesting to note that
`the establishment of
`immortal cell
`lines in vitro is also associated with de novo
`
`methylation of CpG islands [9¢,10]. Other genes knownto
`contribute to tumorigenesis such as the DNA-repair gene
`MLHI {1i], E-cadherin [12]. cyclooxygenase-2 [13.14] and
`estrogen
`receptor
`o
`[15.16,17]
`are
`also
`silenced
`by
`methylation of CpG islands in their upstream promoters.
`Additionally, it appears that resistance to chemotherapeutic
`agents may be mediated by methylation of genes in the
`apoptotic pathway. For example,
`the restoration of Apaf-1
`expression in highly chemoresistant melanoma cell
`lines
`after treatment with 5-aza-2'-deoxycytidine, led to a marked
`enhancement in their sensitivity to adriamycin and a rescue
`of the apoptotic defects associated with Apaf-I silencing
`[18ee].
`If these epigenetic changes could be reversecl, we
`would potentially be able to re-establish antiproliferation,
`differentiation and chemotherapy sensitivity pathways in
`malignant cells.
`
`*To whom correspondence should be addressed
`
`Current Opinion in Investigational Drugs 2002 3(4):627-633
`© PharmaPress Ltd ISSN 1472-4472
`
`in
`changes
`importance of epigenetic
`the
`In recent years
`carcinogenesis has been unfolding. It is now clear that the fifth
`base of the genome. methylcytosine. plays a critical role in the
`control of gene expression during normal development and
`carcinogenesis. Efforts to decrease methylation in neoplasias as a
`therapeutic
`strategy have
`been productive
`in
`hematologic
`malignancies but disappointing in solid tumors. The following is a
`review ofthe clinical experience with the agents 5-azacytidine and
`5-aza-2'-deoxycytidine in solid malignancies and a discussion of
`the difficulties encountered.
`
`Keywords 5-Azacytidine, 5-aza-2-deoxycytidine, clinical trials,
`DNAmethylation, solid tumor
`
`DNA methylation
`Methylation occurs after DNA replication by the addition
`of a methyl group from S-adenosylmethionine (SAM)
`to
`the 5'-position of cytosine residues. Approximately 3 to 4%
`of cytosines in mammalian DNA are methylated. Most of
`the 5-methylcytosine residues in eukaryotic DNA are
`found in the dinucleotide sequence 9'-CpG-3'
`[le]. CpG
`dinucleotides are scarce throughout mammalian DNA,
`except in the so-called CpG islands, where their frequency
`is
`normal
`or
`higher
`than
`expected.
`Spontaneous
`deamination of 5-methylcytosine leads to thymine and
`thus methylated CpG sites are highly mutagenic. In fact,
`although CpG dinucleotides are only found at one-fifth of
`the expected frequency in human DNA. more than 30%of
`all known disease-related point mutations are found at
`these sites [2°,3e].
`
`
`
`
`
`
`
` AOVCUUELUPHUY
`
`
`
`AEOSEADIDIED)|
`
` —
`
`(decitabine;
` 5-aza-2'-deoxycytidine
`and
`5-Azacylidine
`SuperGen Inc; Figure 1) are pyrimidine analogs that result
`from substituting nitrogen at the fifth carbon pesition ofthe
`nucleosides
`cytidine
`and 2'-deoxeytidine,
`respectively.
`Whenthey are incorporated into replicating DNA they form
`a covalent complex with DNA methyltransferase | (DNMTI,
`This raises the question of why the fifth base of the
`responsible for reproducing the methylation patterns in the
`genome
`is maintained. The
`essential
`function
`of
`daughter strands) and deplete the cell of this enzymatic
`methylated cytosine residues appears to be to modily
`activity leading to the synthesis of hypomethylated strands
`protein-DNA interactions and thereby suppress gene
`[19°]. At high concentrations these drugs are cytotoxic but at
`transcription. CpG islands are often located in the
`lowerconcentrations they induce differentiation of ccll lines
`promoter regions of genes and it has been shown that
`(20). It is believed that the changes observedincells treated
`methylation of their cytosine residues effectively switches
`with these drugs are a consequence of their induction of
`off the downstream gene [4ee]. Like genetic mutations, a
`DNA demethylation and ensuing activation of silent genes,
`methylation pattern is
`information that
`is stable and
`although some evidence suggests that covalent binding of
`reproduced with each round of cell division, but unlike
`the enzyme to the drug-substituted DNA is the primary
`genetic mutations,
`if
`can be readily reversed. This
`mechanism of
`drug-induced
`toxicity
`[Zle22e]. Both
`property makes DNA methylation an essential
`tool
`compounds have been tested in clinical trials and found to
`
`during Shortly—afterembryonic development.
`
`
`have significant antitumoractivity.
`fertilization,
`the methylation patterns observed in the
`CELGENE 2003
`CELGENE 2003
`APOTEX v. CELGENE
`APOTEX v. CELGENE
`IPR2023-00512
`IPR2023-00512
`
`jes1Gojo0uQ
`
`

`

`628 Current Opinion in Investigational Drugs 2002 Vol 3 No 4
`
`Figure 1. Structures of 5-azacytidine and 5-aza-2'-deoxycytidine.
`
`5-azacytidine
`
`5-aza-2'-deoxycytidine
`(SuperGen)
`
`
`
`Clinical experience in hematologic diseases
`Both 5-azacytidine and 5-aza-2'-deoxycytidine, at doses
`ranging from 500 to 1500 mg/m’, have proved effective in
`the treatment of relapsed or refractory acute leukemias and
`in the blast crisis of chronic mycloid leukemia. At low doses
`(50
`to
`150 mg/m’)
`they
`have
`shown
`activity
`in
`myelodysplastic syndrome (MDS),
`leading to trilineage
`responses in some patients [23]. A randomized phaseIII trial
`of
`low-dose S-azacytidine administered subcutaneously
`demonstrated a decreased probability of transformation to
`acute myeloid leukemia in high risk MDS patients, an
`improvement
`in quality of
`life and a
`trend towards
`improvedsurvival [24]. There have been several reports of
`increase in fetal hemoglobin in patients with severe [-
`thalassemia and sickle cell anemia treated with the cytidine
`analogs, although progress in non-malignant diseases has
`been hampered by the potential risk of carcinogenicity that
`these drugs carry [22¢,25¢|.
`
`Clinical experiencein solid tumors
`5-Azacytidine
`Clinical trials with 5-azacytidine are summarized in Table 1.
`An initial phase | trial was reported in 1972 by Weiss et al
`[26] using doses of 0.55 to 2.4 mg/kg/dayfor 10 to 15 days.
`Thirteen of thirty treated patients were reported to have had
`a partial response (two ofsix colon cancers. seven of eleven
`breast cancers and two offive melanomas amongst them).
`Remissions commonly occurred early in
`the treatment
`course and lasted an average of 6 weeks. Re-induction of
`remissions was possible in two patients with breast cancer
`who hadrelapses at 8 to 10 weeks. A maintenance regimen
`using 2.4 mg/kg twice a week was given to responding
`patients and two of these remained in remission for at least 6
`months.
`
`Following these encouraging results, several phase II trials
`were conducted. At
`the Mayo Clinic, 29 patients with
`advanced gastrointestinal cancer were treated with 500 to
`750 mg/m’ per course [27]. Nausea and vomiting were so
`severe,
`however,
`that
`three
`different
`administration
`schedules were tried in an attempt
`to decrease
`the
`symptoms. The drug was given once-daily for 5 days, once-
`daily for 10 days and twice-daily for 5 days. The latter was
`the
`better-tolerated
`regimen
`but was_
`still
`seriously
`emetogenic. Only one partial response lasting 5 weeks was
`observed. A cooperative phase II study in 31 patients with
`breast cancer using 60 mg/m‘/day of
`intravenous 5-
`azacytidine for 10 days (followed by a maintenance dose of
`100 mg/m’ twice weekly after bone marrow recovery) [28]
`yielded only four disease stabilizations and two partial
`
`responses lasting a mean of 5.5 months. Another small phase
`Ul trial was reported in 1982 [29], where eight patients with
`osteogenic sarcomas and seven with skeletal Ewing's
`sarcoma were treated with {50 to 200 mg/m’/day every8 h
`for 5 days: no objective responses were seen.
`
`large studies involving a spectrum of solid tumors
`Several
`have been published. The Southwest Oncology Grouptreated
`191
`patients with
`5-azacytidine
`intravenously
`(225
`mg/m’/day)
`for 5 days every 3 weeks [30]. Because of
`myelosuppression, however,
`this dose had to be reduced
`initially to 175 and then to 150 mg/m’. Two patients with
`adenocarcinoma of
`the lung, one with squamous
`cell
`carcinoma ofthe lung and two with embryonal carcinoma of
`the testicle had partial responses lasting from 28 to 77 days.
`Five
`renal, one breast,
`two colon and two_ pancreatic
`adenocarcinomas plus single malignancies arising from six
`other primarysites, were stabilized for 39 to 255 days. 6% Of
`patients died and 11% refused further therapy because ofthe
`drug's severe gastrointestinal toxicity. The Central Oncology
`Group administered 5-azacytidine (1.6 mg/kg/day) for 10
`days to 221 patients [31].
`In an attempt
`to decrease the
`uncontrollable nausea and vomiting induced on the day of
`treatment, 29 patients received the drug as an 18 to 24 h
`infusion. The degrees of leukopenia and thrombocytopenia
`were greater with the slow infusion. Stomatitis as well as an
`erythematous rash appeared, bul nausea and vomiting, were
`minimal. Although 19 partial responses were reported (one
`lung, six breast,
`three lymphoma and nine miscellaneous
`tumors), they were mainly of non-visceral disease and short-
`lived (mean of 5 weeks). The Southeastern Cancer Study
`Group[32] tried a biweekly regimenof 150 mg/m’ (50 mg/m’
`in lymphoma patients) of 5-azacytidine in 91 patients with
`disseminated malignancies, but only obtained two partial
`responses (one large cell carcinoma of the lung and one
`melanoma).
`In all of these trials, all of the patients that
`received the drug as an intravenous bolus suffered severe
`nausea and vomiting that was unresponsive to antiemetics,
`and frequently accompanied by diarrhea. Leukopenia and
`thrombocytopenia were dose-related and occurredlate in the
`course oftreatment.
`
`A small study comparing intravenous and subcutaneous
`administration of 5-azacytidine in humans had shownthat
`plasmalevels were similar after 1 h andthat the drug tended
`to concentrate in tumor tissue regardless of the route used
`{33]. Based on these results, another phase [
`trial was
`conducted
`using
`275
`to
`850 mg/m’
`administered
`subcutaneouslydaily for 10 days followed by 35 to 90 mg/m’
`weekly in those who responded [34].
`18 Patients were
`evaluable for toxicity. Nausea, vomiting and diarrhea were
`mild in this trial, however, severe hepatic toxicity occurred in
`five patients (all with significant hepatic metastatic disease), of
`whichthree died in hepatic coma. The platelet counts ofthree
`patients dropped to < 50,000/mm’, and two died as a direct
`consequence. Only two partial
`responses were observed
`whichlasted 2 and 3 months, respectively.
`
`One study investigated the benefits of administering 5-
`azacytidine with pyrazofurin (PF; an inhibitor of the enzyme
`orotidylate decarboxylase
`in
`the
`de nove pyrimidine
`biosynthesis pathway) after significant synergism ofthese
`drugs had been demonstrated in cell cultures
`[35].
`5-
`Azacytidine was given as a continuous intravenous infusion
`
`

`

`Clinical experience with 5-azacytidine and 5-aza-2'-deoxycytidine in solid tumors Aparicio & Weber 629
`
`Table 1. Clinical trials of 5-azacytidine in solid tumors.
`Reference |
`Objective
`Schedule
`‘Dose
`Study
`Numberof
`patients
`responses
`
`(Evaluable)
`:
`.
`Phase |: Breast, colon, melanoma,
`:
`30.
`0.55 to 2.4 mg/kg lv bolus:
`gd x 10 to 15 days
`43 PR
`[26]
`lung, soft tissue sarcomas, ovary,
`:
`pancreas, lymphoma
`
`Phase Il: Colon, one pancreas
`and_one gastric
`Phase |: Ovary, breast, tonsil,
`lung, hepatoma, renal, colon,
`melanoma, chordoma
`
`Phase |} Breast
`
`Phase |i: Solid tumors’
`
`:
`
`29
`
`18
`
`27
`
`167
`
`500 to 750 mg/miv bolus.
`(total dose per course)
`275 to 850 mg/m’ sc
`(M: 35 to 90 mg/m’)
`
`-qd’x 5 days; qd x 10
`days: bid x 5 days
`qd x 10 days
`(M: once a week)
`
`60 mg/m/day iv bolus
`(M: 400 mg/m’)
`
`gd x 10 days
`(M: biweekly)
`
`150 to 225mgimiv bolus.
`g
`
`qd x 5 days
`every 3 weeks
`
`1 PR
`
`:
`
`2PR
`
`2PR
`4SD
`
`5.PR
`- 16SD
`
`19PR
`
`2PR
`no responses
`See
`no responses
`
`[27]
`
`[34]
`
`[28]
`
`-[30]
`
`:
`
`[34]
`
`[32]
`-[35]
`:
`
`[29]
`
`qd x 10 days
`(M: biweekly)
`Biweekly x 6 days
`qd x 5 days”
`
`Every 8h x 5 days
`
`PhaseII: Solid tumors?
`
`1.6 mg/kg iv bolus/18 to...
`24-h infusion
`(M: 2.4 mg/kg)
`150 mg/miv bolus
`PhaseII: Solid tumors’
`30 to 60 mg/m’
`Phase |: In combination with
`continuous iv infusion
`pyrazofurin
`“Phase ll: Sarcomas of the bone a 150 mg/m’iv over 3 h
`
`177
`
`91
`6
`
`M maintenance, qd oncedaily, PR partial response, SD stable disease.
`lung (adenocarcinoma (12),
`‘Tumor and number of patients shown in parentheses: Pancreas (adenocarcinoma (6) andislet cell carcinoma (1));
`squamouscell carcinoma (14) and undifferentiated cell carcinoma (12)); colorectal (15); kidney (17); breast (14); testicle (embryonalcell carcinoma
`(2), teratocarcinoma (1), choriocarcinoma (1)); urinary bladder (2); primary liver carcinoma (4); palate adenocarcinoma(1); parotid adenocarcinoma
`(3); uterus adenocarcinoma (1); cervix squamouscell carcinoma (4); ovary (5); non-Hodgkin's lymphoma (4); sarcoma (7); melanoma (13); tongue
`squamous cell carcinoma (2); nasal antrum (1); esophagus squamous cell carcinoma (3); stomach adenocarcinoma (7); skin squamous cell
`carcinoma(1); larynx squamouscell carcinoma (1); gall bladder (1); unknown primary (adenocarcinoma (8) and undifferentiated cell carcinoma (4)).
`“Tumor and numberof patients shown in parentheses: Lung (24); breast (29); large intestine (26); melanoma (12); Hodgkin's disease (6);
`non-Hodgkin's lymphoma (8); miscellaneous (59).
`*Tumor and numberof patients shown in parentheses: Breast (6); renal (10); other urogenital (3); colon (7); pancreas (2); stomach (3);
`rectum (1); lung (unspecified (2), squamouscell (6), adenocarcinoma (11), undifferentiated (5), small cell (2), large cell (2)); melanoma (10);
`head and neck(14): soft tissue sarcomas (3); lymphomas(4).
`
`
`for 5 days immeciiately following the injection of PF. PF
`doses ranged from 50 to 100 mg/m’ and5-azacytidine doses
`ranged from 30 to 60 mg/m’. The most commonsideeffect
`was skin rash, which was dose-related, and when severe
`was accompanied by stomatitis, proctitis and cystitis. Six
`patients with solid tumors were enteredinto the trial but no
`objective responses were observed.
`
`5-Aza-2'-deoxycytidine
`Clinical trials with 5-aza-2'-deoxycytidine in solid tumors are
`summarized in Table 2. The first phase | trial conducted with
`3-aza-2'-deoxycytidine used a schedule consisting of three
`consecutive 1-h infusions separated by 7 h [36], a schedule
`dictated by
`the
`instability of 5-aza-2'-deoxycytidine
`in
`aqueous solution andits short half-life. The starting dose was
`25 mg/m’. The dose-limiting toxicity consisted of reversible
`myelosuppression, with the white blood cell count nadir
`delayed to days 22 to 33 of treatment. Platelet nadir was
`observed between days 14 and 22. The maximumtolerated
`dose was 100 mg/m’ x 3. One partial response was observed
`in a patient with a
`locally recurrent undifferentiated
`carcinoma of the ethmoid sinus who continued 5-aza-2'-
`
`deoxycytidine treatment every 5 to 6 weeks and, after surgery
`of a
`residual
`lymph node mcetastatasis, remained free of
`
`the EORTC
`disease at 14 months. Based on these results,
`conducted phase Il
`trials with 5-aza-2'-deoxycytidine in
`patients with melanoma and colorectal, renal and head and
`neck cancers, using the same schedule evaluated in the phase
`I study, at a dose of 75 mg/m’ [37]. Of 82 evaluablepatients,
`only one short-lived partial
`response was observed in a
`patient with malignant melanoma. Tumor stabilization was
`reported in 22%ofthe patients with melanoma, in 15%ofthe
`patients with head and neck cancer,
`in 14%of the patients
`with renal cell carcinoma and in 7% of the patients with
`colorectal carcinoma. Despite the lack ofsignificant activity in
`these studies, the EORTC used the same schedule and dose in
`14 patients with non-seminomatoustesticular cancer, because
`of the postulated activity of 5-aza-2'-deoxycytidine as a
`differentiating agent, but no objective responses were seen
`[38]. The same dose and schedule were also used in three
`other phaseII trials in patients with uterine cervical cancer (n
`= 14), ovarian cancer (n = 21) and prostate cancer (n = 12). No
`responses were seen in the uterine cervical cancer group but
`stabilization of disease was observed in two patients with
`ovarian cancer and twopatients with prostate cancer [39-41].
`The most common non-hematologic toxicity encounteredin
`all
`trials with this regimen was mild-to-moderate nausea
`and vomiting.
`
`uy
`
`fe)
`3ito
`QQ
`
`

`

`630 Current Opinion in investigational Drugs 2002 Vol 3 No 4
`
`Table 2. Clinicaltrials of 5-aza-2'-deoxycytidine in solid tumors.
`Objective
`Study
`Numberof
`Dose
`patients
`responses
`
`_(Evaluabie)
`i
`Phase|
`i
`20
`25 to 100 mg/m’
`:
`Every 3 to 6 weeks
`4.PR
`{36}
`:
`over Th every 8hx2te3
`
`Reference
`
`Schedule
`
`82
`s
`
`i. mg/m’ over 4h.
`every 8hAX3-
`
`Every 5 weeks
`
`1PRand13SD.
`:
`
`[87]
`
`Phase Il: Melanoma,colorectal,
`renal and squamouscell cancer
`of the head and neck
`Phase Il: Ovarian cancer
`e
`PhaseIl: Cancerofthe uterine
`cervix
`"PhaseIl:Non-seminomatous|
`testicular cancer
`Phase I/Il: Non-small cell fang :
`cancer
`Phase ll: Prostate cancer:
`¥:
`:
`;
`Phase
`
`Phase |I: Non-small cell jung
`cancer
`ins
`Phase
`
`
`
`2d
`
`14
`
`e
`
`:
`
`ce 14
`:
`9
`12
`
`21
`
`14
`19
`
`75 mg/m’ over1 A
`every 8h x3
`75 mgimover 4 he
`every 8h x3-
`75 mgimover 1h.
`every 8h x3
`:
`200 to 660 maim! x1
`over 8h
`75 inantover 1 h
`every 8hx3_
`45 to 120 mg/n*
`over 1h +33 mg/m’
`cisplatin every 24h x 3
`OL mit’ over th + 33
`“mg/mcisplatin every 24
`hx3
`20to40 atid
`: “over 72h
`
`
`Every 5 weeks
`:
`Every 6.weeks
`:
`
`Every 5'weeks
`j
`Every & to 7weeks
`oS
`‘Every 5 to8 weeks
`:
`‘
`Every 3 weeks
`
`:
`
`28D
`
`:
`
`no responses
`:
`no responses.
`:
`SA SD nt:
`28D
`
`2
`
`4PRand2
`minor responses
`
`Every 3 weeks
`_EverySweeks
`
`:
`
`3 minor
`responses
`shoresponses
`
`[40]
`
`[39]
`:
`[38]
`:
`{43}
`44]
`S
`
`47]
`
`:
`
`[47]
`polddle
`
`cs
`
`cell lung cancer used 5-aza-2'-deoxycytidine (67 mg/m’)
`the
`Both in vitro and in vive data [42] suggested that
`andcisplatin (33 mg/m’) on days 1 to 3. Only three short-
`cytotoxicity of 5-aza-2'-deoxycytidine was dose- and time-
`
`dependent. schedules—ofTherefore, alternative
`
`
`lasting minor regressions were observed[47].
`administration were explored.
`In 1997 Momparler et al
`conducted a phase I/II
`trial
`in previously untreated
`patients with stage IV non-small cell lung cancer, using an
`8-h intravenous infusion of 5-aza-2'-deoxycytidine at
`a
`dose of 200 to 660 mg/m’. They reported an increase in
`survival time with the number of cycles administered and
`one long-term survivor. They concluded that 5-aza-2’-
`deoxycytidine has a delayed action on tumor growth that
`may require several cycles of treatment before becoming
`evident |43]. Our group conducted a phase [ trial using a
`72-h continuous intravenous infusion of doses between 20
`and 40 mg/m’/day. All of
`the patients were heavily
`pretreated and only one ofthe 19 wasable to receive more
`than one cycle of treatment. No objective tumor responses
`were observed [44].
`
`Detection of DNA methylation
`in DNA
`To clinically assess
`the utility of alterations
`methylation,
`it
`is
`important
`to accurately measure the
`changes in methylation that occur after treatment with
`demethylating agents. Until
`recently,
`the
`techniques
`available for this have been difficult
`to apply to clinical
`samples.
`
`synergistic
`demonstrated
`have
`studies
`vitro
`In
`cytotoxicity using 5-aza-2'-deoxycytidine and cisplatin, 4-
`hydroperoxycyclophosphamide
`{a
`derivative
`of
`cyclophosphamide) and topotecan [45,46]. Based on these
`results, a phase | trial was conducted by Schwartsmannet
`al
`[47]
`using cisplatin
`(33 mg/m‘)
`plus
`5-aza-2’-
`deoxycytidine (45 to 120 mg/m’) as a 2-h infusion on
`days 1
`to 3. One partial response was observed in a
`patient with advanced cervical cancer. A follow-up early
`phase II evaluation in 14 patients with inoperable non-small
`
`Initial protocols to detect changes in DNA methylation
`employed digestion of genomic DNA with methylation-
`sensitive restriction enzymes (characterized bytheir inability
`to
`cleave
`sequences
`that
`contain methylated CpG
`dinucleotides) followed by Southern blot analysis [le], The
`main drawbacks to this method included a requirement for
`large amounts of DNA (> 5 ug) and that
`the extent of
`analysis was limited to the CpG sites present
`in the
`recognition sites of the available enzymes. Later on, PCR
`amplification was applied using primers that flanked the
`restriction sites that were methylation sensitive. Although
`the sensitivity of the assay increased, this method could still
`only be used to assess CpG methylation at methylation-
`sensitive restricion sites, and it had the potential of
`generating false positive results if
`the cleavage of
`the
`unmethylated DNA was incomplete [48].
`
`

`

`asosyuy|
`
`
`
`
`
`
`
` _Aomuueyurnuy|
`
`Clinical experience with 5-azacytidine and 5-aza-2'-deoxycytidine in solid tumors Aparicio & Weber 631
`
`then
`bisulfite conversion of the sample DNA, which is
`amplified
`by
`PCR using
`three methylation-specific
`oligonucleotides: a probe with a 5'-fluorescent reporter dye
`and a 3'-quencher dye, and two locus specific PCR primers
`that flank the probe. During the PCR reaction, the nuclease
`activity of the Tag DNA polymerase cleaves the probe and
`the reporter is released resulting in a fluorescent signal that
`is proportional to the amount of PCR product generated and
`immeasurable with a real-time fluorescence instrumentation.
`
`This technology has mace possible the rapid screening of
`large numbers of human tumors for the methylation state of
`a parlicular locus [53] but cannot offer information on longer
`regions of DNA.
`
`technique to analyze the biological activity of
`The ideal
`demethylating agents in clinical
`trials should be able to
`combine the high-resolution information of the methylation
`state of a
`large area of DNA sequence,
`like bisulfite
`sequencing. with the sensitivity, accuracy and efficiency of
`high-throughput technology.
`
`5-
`detect
`to
`used
`also
`sequencing was
`Genomic
`methylcytosine residues, identified as a lack of bands in all
`tracks of a sequencing gel. However,
`interpretation was
`frequently complicated by close spacing of the bands or by
`backgroundcleavage lacders [te]. In 1992, Frommer ef al
`[49] described the bisulfite genomic sequencing technique
`based on treatment of single stranded DNA with sodium
`bisulfite to deaminate cytosine to uracil muchfaster than 5-
`methylcytosine to thymine,
`so that methylated cytosine
`residues are left
`intact. The bisulfite-treated DNA was
`amplified by PCR and the products sequenced. The 5-
`methylcytosine residues on the original sample appeared as
`the only remaining cytosines on the sequencing gel, since
`unmethylated cytosines were transformed to uracil by the
`bisulfite. This approach could be applied to small amounts
`of DNA; even DNA obtained from paraffin-embedded
`tissue samples. However, without cloning the amplified
`roducts, a labor-intensive and time-consuming process,
`his method was less sensitive than Southern analysis.
`evertheless, bisulfite treatment of genomic DNA provided
`he basis for multiple new strategies for the assessment of
`DNA methylation.
`
`
`
`
`
`References
`
`th
`
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`[52] developed a high-throughput
`et al
`Finally, Eads
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`
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`Future directions
`role in the
`Since DNA methylation plays an important
`[50] described the methylation-specific PCR
`Herman et al
`expression,
`
`
`regulation of—gene modulating it
`
`protocol in which they used three sets of primers designed
`pharmacologically is a very attractive therapeutic target. 5-
`specifically to amplify three types of bisulfite-treated DNA:
`Azacytidine
`and
`5-aza-2'-deoxycytidine have not
`been
`methylated, unmethylated and DNA that had not been
`dramatically successful, and pose significant restriclions on
`modified,
`The
`PCR
`products were
`compared
`on
`dosing schedule adjustments because of their instability in
`polyacrylamide
`gels
`stained with
`ethidium bromice,
`aqueous solution andtheir side effects. Development of new
`providing semiquantitative results. This method was highly
`drugs without Uvese limitations is a subject of active research.
`sensitive, capable of detecting even 0.1%of methylated DNA
`Other approaches to inhibit DNMTI. the enzymeresponsible
`in a sample, and significantly less time consuming than
`for the replication of the DNA methylation pattern, are being
`genomic sequencing. Gonzalgo and Jones [48] developedthe
`explored, incluciing the use of modified oligonucleotides that
`methylation-sensitive single nucleotide primer extension
`directly
`anlagonize
`the
`enzyme
`[54].
`Antisense
`assay in which the PCR product from the bisulfite-convertec
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`DNA amplification was isolated from an agarose gel andused
`clinfeal
`trials have moved ahead [56e]. Combinalions of
`as a template for a second PCR reaction. The second PCR
`demethylating agents with synergistic drugs, such as the
`reaction utilized “P-labeled dNTPs and internal primers tha
`histone deacetylase inhibitors (trichostatin A [57] and 4-
`terminated immediately 5' of the single nucleotide ofinterest.
`phenylbutyrate [58]), or targeting specific signal transduction
`The radiolabeled products were electrophoresed resulting in
`pathwaysthat rendercells sensitive to chemotherapy agents,
`two bands: the C band represented the methylated cytosine
`are other strategies that shouldbe further investigated.
`residues and the T band represented the unmethylatec
`cytosine
`residues. Phosphorimage
`analysis
`allowed for
`quantitation of each band. At the same time, Xiong and Laird
`[51] reported a combinedbisulfite restriction analysis,
`that
`involved a standard sodiumbisulfite PCR treatment, followec
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`the purified PCR products using a
`restriction enzyme with CpG in its recognition sequence. In
`this way, cleavage only occurred if the CpG sequence had
`been retained during the bisulfite conversion, ie, if the origina
`C residue was methylated. Different
`restriction enzymes
`detected different levels of DNA methylation, depending on
`the number of CpG dinucleotides containedin its recognitior
`sequence. Gel
`electrophoresis,
`oligo hybridization anc
`phosphorimage analysis allowed quantitation of the level of
`methylation present. Although these methods were rapid,
`offered quantitative results and were compatible with paraffin
`4.
`Jones PA, Laird PW: Cancer epigenetics comes of age. Nat Genet
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`
`
`embedded they—stilltissues, required cumbersome
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`(1999) 24:163-167.
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`ee This paper reviews the mechanisms of DNA methylation in malignant
`transformation.
`numbers of specimens.
`
`
`
`
`
`leveesia)
`
`flex)
`
`

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