Department of Pharmacology and Therapeutics
Roswell Park Cancer Institute
Elm and Carlton Streets
Buffalo NY USA 14263
Tel: 716 - 845 - 3443
Fax: 716 - 845 - 1575
E-mail: terry.beerman@roswellpark.org

Programs

Cellular response to enediyne induced DNA double strand breaks:

Cellular responses to ionizing radiation (IR)-induced DNA double strand breaks (DSBs) involve activation, via the phosphatidylinositol 3-kinase-like kinase (PIKK) ATM, of cell cycle checkpoint proteins that delay cell cycle progression to allow DNA repair and to preserve chromosomal integrity. According to this model, IR-induced breaks are repaired by two major DNA repair pathways: non-homologous end-joining (NHEJ), which processes breaks in G1 and early S, and homologous recombination (HR), which acts primarily on damage induced during late S and G2.Thus, in IR-treated cells, activation of checkpoints helps ensure that cells entering mitosis contain minimal chromosomal aberrations.

We have accumulated evidence that responses to DNA DSBs induced by the radiomimetic enediyne C-1027 and its family members deviate from this model. For example, IR induces ATM-dependent, and C-1027, both ATM-independent, responses. Furthermore, treatment of cells with equi-toxic levels of IR or C-1027 results in either a very low (2.9%), or an extraordinarily high (92%), percentage, respectively of mitotic cells showing aberrant chromosomal recombination. FISH analysis revealed that C-1027-induced chromosomal aberrations are frequently associated with disruption of telomeres.

Defining the mechanism(s) behind these unique responses to C-1027 should help extend our understanding of DNA damage responses to DSBs. First, pivotal checkpoint pathway proteins involved in the response to C-1027 and several recently available analogs will be identified, with emphasis on both ATM-dependent and independent responses. Also, the conditions under which C-1027 induces extensive chromosomal fusions, characterized by aberrant end-joining and fragmentation, will be identified. We, will test whether C-1027, which induces DSBs preferentially within a GTTA motif, targets the telomere tandem repeat sequence (GGGTTA) and contributes to aberrant end-joining by inducing telomere dysfunction. Selected C-1027 analogs with cleavage characteristics different from C-1027 will be tested to provide additional mechanistic insights into which properties of C-1027-induced damage dictate the DNA damage response.

Cellular response to drug induced stalling of DNA replication:

This study will identify replication arrest/recovery mechanism(s) stemming from DNA damage induced by the highly cytotoxic monofunctional alkylators adozelesin, hedamycin and Et743. These drugs both alkylate DNA and induce helical changes and are potent inhibitors of DNA replication. Stalled DNA replication, even when induced by different types of DNA damaging treatments (e. g., mitomycin C, ultraviolet light and camptothecin), is thought to invoke a common DNA damage response. However, recent studies from this laboratory revealed varied DNA damage responses stemming from replication arrest induced by each of the listed drugs including:First, equi-cytotoxic concentrations of these alkylators induce markedly different cell cycle checkpoint respnses, precluding a common DNA damage response. Second, while all are highly potent inhibitors of DNA replication, each agent appears to stall replication by a different mechanism. Third, preliminary data suggest that the alkylator-induced cellular responses are accompanied by varying amounts of interphase nuclear DNA breaks and associated metaphase chromosomal aberrations (i. e., chromosome fragmentation and recombination events). Third, preliminary data suggested that the alkylator-induced cellular responses are accompanied by varying amounts of interpahse nuclear DNA breaks and associated metaphase chromosomal abberations (i.e., chromosome fragmentation and recombination events).Thus, cellular responses to replication stalling by these drugs appear to be agent specific.

We will identify replication arrest and recovery mechanisms associated with each of the selected alkylators. Mechanisms by which the alkylators stall DNA replication (i. e., whether inhibition occurs in trans and/or in cis, and if inhibition is at the level of initiation and/or elongation) will be determined. Relationships between replication arrest and DNA breaks/recombination events and/or chromosomal aberrations will be characterized. Whether recovery from alkylator-induced replication stalling involves the action of specialized bypass polymerases to advance past stalled replication sites will be tested.

Radiomimetics: Cells lacking the protein kinase ataxia telangiectasia mutated (ATM) have defective responses to DNA double strand breaks (DSBs) including an inability to activate damage response proteins such as p53. However, we previously showed that cells lacking ATM robustly activate p53 in response to DNA strand breaks induced by the radiomimetic enediyne C-1027. To gain insight into the nature of C-1027-induced ATM-independent damage responses to DNA DSBs, we further examined the molecular mechanisms underlying the cellular response to this unique radiomimetic agent. Similar to ionizing radiation (IR) and other radiomimetics, breaks induced by C-1027 efficiently activate ATM by phosphorylation at Ser1981. Yet unlike other radiomimetics and IR, DNA breaks induced by C-1027 results in normal phosphorylation of p53 and the cell cycle checkpoint kinases (Chk1 and Chk2) in the absence of ATM. In the presence of ATM, but under ATM and Rad3-related kinase (ATR) deficient conditions, C-1027 treatment resulted in a decrease of Chk1 phosphorylation but not in p53 and Chk2 phosphorylation. Only when cells were deficient in both ATM and ATR, was there a reduction in phosphorylation of each of these DNA damage response proteins. This reduction was also accompanied by increased cell death in comparison to wild-type cells or cells lacking either ATM or ATR. Our findings demonstrate a unique cellular response to C-1027-induced DNA DSBs in that DNA damage response proteins are unaffected by the absence of ATM, as long as ATR is present.

Radiomimetics: The radiomimetic enediyne C-1027 induces almost exclusively DNA double strand breaks (DSBs) and is extremely cytotoxic. Uniquely amongst radiomimetics, ATM is dispensable for cellular responses to C-1027-induced DNA damage. This study explores the biological activity of three recently bio-engineered C-1027 analogs, 7"-desmethyl-C-1027 (desmethyl), 20'-deschloro-C-1027 (deschloro), and 22'-deshydroxy-C-1027 (deshydroxy). Each compound maintains the characteristic ability of radiomimetics to cleave DNA in cell-free systems, varying in activity from 3 (deschloro) to 40 fold (desmethyl) less than C-1027. The induction of cellular DNA breaks, based upon pulsed field gel electrophoresis, comet analysis and H2AX activation, was in the same rank order as cell-free DNA break induction, although the amount of breaks induced by desmethyl is greatly reduced compared to the other analogs. Despite the disparity in inducing DNA DSBs, all of the analogs produced G₂/M cell cycle arrest and activated DNA DSB damage response proteins such as p53-Ser15 and Chk2-Thr68 at concentrations in concordance with their ability to inhibit cell growth. Interestingly, of the three analogs, only the desmethyl-induced DNA damage response was similar to C-1027, as it did not cause hypersensitive cell growth inhibition in the absence of ATM nor require the kinase to phosphorylate p53 or Chk2. These findings demonstrate that simple modifications of C-1027's chromophore can result in varied induction of, and cellular response to, DNA DSBs.

Radiomimetics: The ability of the radiomimetic anticancer enediyne C-1027 to induce novel ataxia-telangiectasia mutated (ATM) or ATM-and-Rad3-related (ATR) independent damage responses was discovered to reside in its unique ability to concurrently generate robust amounts of DNA double strand breaks (DSBs) and interstrand crosslinks (ICLs) in cellular DNA. Furthermore, a single substitution to the chromophore's benzoxazolinate moiety shifted DNA damage to primarily ICLs and an ATR, but not ATM dependent, DNA damage response. In contrast, single substitutions of the chromophore's β-amino acid component shifted DNA damage to DSBs, consistent with its induction of conventional ATM-dependent DNA damage responses of the type generated by ionizing radiation (IR) and other radiomimetics. Thus, phosphatidylinositol 3-kinase-like protein kinase (PIKK) regulation of responses to DNA damage is dictated by the relative proportions of DSBs and ICLs.

Replication stalling: Et743, is a highly cytotoxic anticancer agent isolated from the squirt Ecteinascidia turbinate, which alkylates DNA in the minor groove at GC rich sequences resulting in an unusual bending towards the major groove. Et743's ability to block DNA replication was studied using the well-established SV40 intracellular and cell-free model of mammalian DNA replication. Low concentrations (e.g. 30-100 nM) of Et743 rapidly inhibited elongation and initiation of intracellular SV40 DNA synthesis in virally infected BSC-1 cells. Despite inducing a low frequency of adducts on the DNA template, these concentrations caused a substantial decrease in intracellular SV40 DNA replication activity indicating trans-acting inhibition. However, extracts of cells treated with the same concentrations of Et743 were fully competent to support cell-free replication, indicating that neither replication factors were depleted nor was an active inhibitor of replication induced. Treatment of SV40 DNA replication templates with Et743 resulted in reduced replication activity only at high adduct frequency, suggesting that drug adducts are not efficient barriers to DNA replication.

Comparative studies involving other DNA alkylators, tomamycin and saframycin A, revealed a decrease in intracellular SV40 replication inhibition at concentrations approximately 10 times higher than Et743, while tomamycin- or saframycin A-adducted DNA templates inhibited cell-free replication within similarly to Et743. Et743 appears to be unusual among other alkylators, as its adducts strongly inhibit intracellular SV40 DNA replication but are relatively weak as cis inhibitors of cell-free DNA synthesis.

Replication stalling: Hedamycin, is a monofunctional DNA alkylator of the pluramycin class of antitumor antibiotics, Hedamycin is highly cytotoxic, and at a concentration of 200 pM decreased growth of mammalian cells by 50%. At hedamycin concentrations similar to those required to interfere with cell growth, DNA synthesis was rapidly decreased, while substantially higher levels were needed to block RNA synthesis. Protein synthesis, even at very high hedamycin concentrations remained unaffected. In contrast to other highly cytotoxic monofunctional DNA alkylators like adozelesin and Et743, hedamycin did not significantly block cells in G2 or increase the sub G1 cell population at higher concentrations. Rather, hedamycin caused substantial S-phase arrest, and, at increasing concentrations, complete cell cycle arrest. Neither inhibition of cell growth or S-phase arrest appeared to be dependent upon ATR expression. Western analysis demonstrated that by 24 h under treatment conditions where hedamycin began to interfere with S-phase progression, there were substantial increases in p53Ser15, chk1 and chk2 phosphorylation as well as loss of cdc25A. Additionally, under conditions where a significant percentage of cells were blocked in S-phase, increases were observed for E2F1, cyclin E and p21 expression, responses associated with halting initiation of DNA replication. Changes in DNA damage response proteins such as chk1, chk2, p53Ser15, and cdc25A were rapid and could be seen within 15-30 min following hedamycin treatment. Under conditions of substantial S-phase arrest, several indicators of apoptotic events including positive TUNEL staining and PARP cleavage were indicated although no change was noted in the sub G1 cell population. However, the apoptotic markers were significantly reduced at higher hedamycin concentrations which prevented cells from leaving G1. Under these conditions, levels of phosphorylated chk1 and chk2, E2F1, cyclin E and p21 also diminished. Cellular responses to hedamycin are unique in comparison to several other highly cytotoxic monofunctional alkylators in that moderate concentrations induce a predominant block in S-phase progression, while at higher concentrations cell cycling is blocked in G1 along with a decrease in apoptotic associated activities.

Laboratory Personnel:

• Terry Beerman Mary McHugh (Senior Invest)
• Loretta Gawron (Tech)
• Jeff LaDuca (Tech part time)
• Michelle Shin (Student)
• Christine Collins (Student)

RPCI and U. of Buffalo Collaborators:

• Sei-ichi Matsui
• Dave Kowalski
• Tom Melendy
• Piero Bianco

Key Publications

Mary M. McHugh, Loretta S. Gawron, Sei-Ichi Matsui and Terry A. Beerman. The DNA damaging enediyne C-1027 induces chromosomal aberrations, altered cell cycle progression and telomere dysfunction. Cancer Research, 65:5344-5351, 2005.

Lan Chun Tu, Sei ichi Matsui, and Terry A. Beerman. The monofunctional DNA alkylator hedamycin induces DNA breaks and chromosome aberrations leading to H2AX activation: involvement of PIKKs and DNA replication fork movement. Molecular Cancer Therapeutics,4:1175-1185, 2005.

Daniel Kennedy and Terry Beerman. The radiomimetic enediyne C-1027 induces unusual DNA damage responses to double strand breaks. Biochemistry, 45:3747-3754, 2006.

Barbara Dziegielewska, Terry Beerman and Piero Bianco. Inhibition of RecBCD enzyme by antineoplastic DNA alkylating agents. J. Molec. Biology, 361: 898-919, 2006.

Daniel Kennedy, Loretta Gawron, Jianhua Ju, Wen Liu, Ben Shen and Terry Beerman. Single chemical modifications of the enediyne core of C-1027, a radiomimetic antitumor drug, affect both drug potency and the role of Ataxia-Telangiectasia Mutated in cellular responses to DNA double strand breaks. Cancer Research, 67: 773-781, 2007.