DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells.

Although autophagy enhances cell survival in nutrient-deprived cells by increasing adenosine triphosphate (ATP) production, it remains unclear if autophagy functions similarly in cells treated with cytotoxic chemotherapy agents. To address this issue, we measured both the ability of DNA damaging agents (Temozolomide, and Etoposide) to induce an autophagy-dependent production of ATP, and the effects of modulation of autophagy on drug-induced cell death. Both drugs induced an autophagy-associated increase in ATP production in multiple glioma cell lines. The drug-induced ATP surge could not be blocked by glucose starvation, but could be blocked by preincubation with the autophagy inhibitor 3-methyladenine (3-MA), an siRNA targeting beclin 1, or the mitochondrial inhibitor oligomycin. Inhibition of autophagy-induced ATP production increased non-apoptotic cell death associated with micronucleation, while restoration of the 3-MA-inhibited ATP surge by addition of pyruvate suppressed cell death. These results show that DNA damaging agents induce an autophagy-associated ATP surge that protects cells and may contribute to drug resistance.

Aberrant silencing of the CpG island-containing human O6-methylguanine DNA methyltransferase gene is associated with the loss of nucleosome-like positioning.

Tumor-associated aberrant silencing of CpG island-containing genes has been correlated with increased cytosine methylation, a "closed" chromatin structure, and exclusion of transcription factor binding in the CpG island/promoter regions of affected genes. Given the lack of understanding of what constitutes a closed chromatin structure in CpG islands, however, it has been difficult to assess the relationship among cytosine methylation, chromatin structure, and inappropriate gene silencing. In this study, nuclease accessibility analysis was used to more clearly define the chromatin structure in the CpG island of the human O6-methylguanine DNA methyltransferase (MGMT) gene. Chromatin structure was then related to in vivo DNA-protein interactions and cytosine methylation status of the MGMT CpG island in human glioma cells varying in MGMT expression. The results of these studies indicated that the "open" chromatin structure associated with the MGMT CpG island in MGMT+ cells consisted of an approximately 250-bp transcription factor-binding, nuclease-accessible, nucleosome-free region of DNA, whose formation was associated with at least four flanking, precisely positioned nucleosome-like structures. In MGMT- cells, this precise nucleosomal array was lost and was replaced by randomly positioned nucleosomes (i.e., the closed chromatin structure), regardless of whether methylation of the CpG island was spread over the entire island or limited to regions outside the transcription factor binding region. These results suggest that CpG islands facilitate the expression of housekeeping genes by facilitating nucleosomal positioning and that the conditions that alter the formation of this array (such as perhaps methylation) may indirectly affect CpG island-containing gene expression.

Methylation-related chromatin structure is associated with exclusion of transcription factors from and suppressed expression of the O-6-methylguanine DNA methyltransferase gene in human glioma cell lines.

There is considerable interest in identifying factors responsible for expression of the O-6-methylguanine DNA methyltransferase (MGMT) gene, as MGMT is a major determinant in the response of glioma cells to the chemotherapeutic agent 1,3 bis(2-chloroethyl)-1-nitrosourea. Recently we have shown that MGMT expression is correlated in a direct, graded fashion with methylation in the body of the MGMT gene and in an inverse, graded fashion with promoter methylation in human glioma cell lines. To determine if promoter methylation is an important component of MGMT expression, this study addressed the complex interactions between methylation, chromatin structure, and in vivo transcription factor occupancy in the MGMT promoter of glioma cell lines with different levels of MGMT expression. Our results show that the basal promoter in MGMT-expressing glioma cell lines, which is 100% unmethylated, was very accessible to restriction enzymes at all sites tested, suggesting that this region may be nucleosome free. The basal promoter in glioma cells with minimal MGMT expression, however, which is 75% unmethylated, was much less accessible, and the basal promoter in nonexpressing cells, which is 50% unmethylated, was entirely inaccessible to restriction enzymes. Despite the presence of the relevant transcription factors in all cell lines examined, in vivo footprinting showed DNA-protein interactions at six Sp1 binding sites and one novel binding site in MGMT-expressing cell lines but no such interactions in nonexpressors. We conclude that in contrast to findings of previous in vitro studies, Sp1 is an important component of MGMT transcription. These correlations also strongly suggest that methylation and chromatin structure, by determining whether Sp1 and other transcription factors can access the MGMT promoter, set the transcriptional state of the MGMT gene.

Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene.

O6-Methylguanine DNA methyltransferase (MGMT) repairs the mutagenic and cytotoxic O6-alkylguanine lesions produced by environmental carcinogens and the chemotherapeutic nitrosoureas. As such, MGMT-mediated repair of O6-alkylguanine lesions constitutes a major form of resistance to nitrosourea chemotherapy and makes control of MGMT expression of clinical interest. The variability of expression in cell lines and tissues, along with the ease with which the MGMT phenotype reverts under various conditions, suggests that MGMT is under epigenetic control. One such epigenetic mechanism, 5-methylation of cytosines, has been linked to MGMT expression. We have used an isogenic human multiple myeloma tumor cell line model composed of an MGMT-positive parent cell line, RPMI 8226/S, and its MGMT-negative variant, termed 8226/V, to study the control of MGMT expression. The loss of MGMT activity in 8226/V was found to be due to the loss of detectable MGMT gene expression. Bisulfite sequencing of the MGMT CpG island promoter revealed large increases in the levels of CpG methylation within discrete regions of the 8226/V MGMT CpG island compared to those in 8226/S. These changes in CpG methylation are associated with local heterochromatinization of the 8226/V MGMT transcription start site and provide a likely mechanism for the loss of MGMT transcription in 8226/V.

UBE2S, a novel substrate of Akt1, associates with Ku70 and regulates DNA repair and glioblastoma multiforme resistance to chemotherapy.

Glioblastoma multiforme (GBM) is the most common primary malignant brain cancer in adults. However, the molecular events underlying carcinogenesis and their interplay remain elusive. Here, we report that the stability of Ubiquitin-conjugating enzyme E2S (UBE2S) is regulated by the PTEN/Akt pathway and that its degradation depends on the ubiquitin-proteasome system. Mechanistically, Akt1 physically interacted with and phosphorylated UBE2S at Thr 152, enhancing its stability by inhibiting proteasomal degradation. Additionally, accumulated UBE2S was found to be associated with the components of the non-homologous end-joining (NHEJ) complex and participated in the NHEJ-mediated DNA repair process. The association of Ku70 with UBE2S was enhanced, and the complex was recruited to double-stranded break (DSB) sites in response to etoposide treatment. Furthermore, knockdown of UBE2S expression inhibited NHEJ-mediated DSB repair and rendered glioblastoma cells more sensitive to chemotherapy. Overall, our findings provide a novel drug target that may serve as the rationale for the development of a new therapeutic approach.

Presetting of chromatin structure and transcription factor binding poise the human GADD45 gene for rapid transcriptional up-regulation.

GADD45 has been suggested to coordinate cell cycle regulation with the repair of DNA damage following ionizing radiation (IR). Although the GADD45 gene is transcriptionally up-regulated in response to IR, alterations in in vivo transcription factor (TF) binding or chromatin structure associated with up-regulation have not been defined. To understand how chromatin structure might influence TF binding and GADD45 up-regulation, key regulatory regions of the gene were identified by in vivo DNase I hypersensitivity (HS) analysis. Chromatin structure and in vivo TF binding in these regions were subsequently monitored in both non-irradiated and irradiated human ML-1 cells. In non-irradiated cells expressing basal levels of GADD45, the gene exhibited a highly organized chromatin structure with distinctly positioned nucleosomes. Also identified in non-irradiated cells were DNA-protein interactions at octamer binding motifs and a CCAAT box in the promoter and at consensus binding sites for AP-1 and p53 within intron 3. Upon irradiation and a subsequent 15-fold increase in GADD45 mRNA levels, neither the chromatin structure nor the pattern of TF binding in key regulatory regions was altered. These results suggest that the GADD45 gene is poised for up-regulation and can be rapidly induced independent of gross changes in chromatin structure or TF binding.

Confluence-induced alterations in CpG island methylation in cultured normal human fibroblasts.

Growth constraint of bacterial and human cells has been shown to trigger genetic mutation. We questioned whether growth constraint might also trigger epigenetic mutation in the form of CpG island methylation. Logarithmically growing normal human fibro-blasts (NHF) displayed little (0-15%) CpG methylation in select regions of three CpG islands [estrogen receptor (ER), E-cadherin (ECAD) and O (6)-methylguanine-DNA methyltransferase (MGMT)] examined. NHF grown to and left at confluence for 2-21 days showed little (<10%) CpG methylation in the ER and ECAD CpG islands. These confluent, growth-arrested cells, however, displayed extensive ( approximately 50%) methylation of the MGMT CpG island. CpG methylation in the MGMT CpG island was not associated with cellular senescence. The methylation was, however, heritable, but not permanent, as the level of CpG methylation in the MGMT CpG island of cells 4 population doublings following replating after confluence were no different from those in confluent cultures, but returned to levels noted in logarithmically growing cells by 10 population doublings following replating. These results suggest that growth constraint can trigger transient epigenetic change even in normal non-senescent human cells.

DNA-directed actions of 3-deazaguanine: effects on DNA integrity and DNA elongation/ligation.

The cytotoxic action of the guanine analogue, 3-deazaguanine, was shown previously to be closely associated with deazaguanine-induced inhibition of DNA synthesis and incorporation of deazaguanine into DNA. The DNA-directed effects of the compound have been further investigated by studying the effect of deazaguanine on DNA integrity, and on the ability of pulse-labeled L1210 cells to synthesize full length DNA. Deazaguanine caused DNA single strand breaks in newly synthesized DNA but not in preformed DNA. The amount of DNA single strand breaks correlated with both deazaguanine exposure and with the amount of deazaguanine incorporated into the DNA. When cells were allowed to recover in drug-free medium for 12 or 24 h after drug exposure little effect on either the amount of DNA single strand breaks or cell viability relative to controls was observed. Deazaguanine also inhibited the ability of L1210 cells to synthesize full length DNA after pulse labeling of DNA. This effect was temporally related to the inhibition by deazaguanine of total DNA synthesis.

Correlation of biochemical effects and incorporation of 3-deazaguanine into nucleic acids to cytotoxicity in L1210 cells.

3-Deazaguanine, a tumor-inhibitory purine antimetabolite, is cytotoxic to L1210 leukemic cells in culture. The log percentage of viability correlated strongly (r2 = 0.986) with the product of the concentration of 3-deazaguanine, or [3-deazaguanine], and period of exposure (t) when [3-deazaguanine] was between 3 and 50 microM, and t was 12 or 24 h. We wished to relate this cytotoxicity to biochemical effects mediated by 3-deazaguanine. 3-Deazaguanine inhibited both DNA and protein synthesis, and both log DNA synthesis and log protein synthesis correlated inversely with [3-deazaguanine] X t and directly with cell viability (P less than 0.001). L1210 cells accumulated 3-deazaguanine 5'-triphosphate to a level of 1.5 nmol/10(6) cells. 3-Deazaguanine treatment had no effect on intracellular cytidine 5'-triphosphate levels, but reduced adenosine 5'-triphosphate and uridine 5'-triphosphate levels by 40% relative to control and guanosine 5'-triphosphate levels by 85% relative to control at a [3-deazaguanine] X t value at which 3-deazaguanine 5'-triphosphate accumulation was near maximal. Incorporation of 2-14C-labeled 3-deazaguanine into DNA and RNA, separated by Cs2SO4 density gradient centrifugation, was demonstrated. Incorporation into DNA was linear versus [3-deazaguanine] X t and correlated inversely with cell viability (P less than 0.001). These data suggest that 3-deazaguanine is anabolized and incorporated into DNA, and that this incorporation is related to decreased DNA synthesis and cell death. The decrease in protein synthesis and diminution of guanosine 5'-triphosphate levels following drug treatment may also contribute to the growth-inhibitory actions of 3-deazaguanine.

p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells.

Temozolomide (TMZ) is a DNA-methylating agent that has recently been introduced into Phase II and III trials for the treatment of gliomas. TMZ produces O6-methylguanine in DNA, which mispairs with thymine during the next cycle of DNA replication. Subsequent futile cycles of DNA mismatch repair can lead to a p53-associated apoptotic cell death, although this mechanism has been described mostly in hematopoietic neoplasms. We studied the action of TMZ in gliomas and the role p53 might play by using U87 glioma cells that were either p53-wild-type or p53-deficient (by virtue of expression of the viral oncoprotein E6). LN-Z308 cells, in which p53 gene is deleted, were also used. p53-proficient U87 MG cells underwent a prolonged, p53- and p21(Waf1/Cip1)-associated G2-M arrest beginning 2 days after TMZ treatment. Although very few of these cells underwent apoptosis, most underwent senescence over a 10-day period. p53-deficient (E6-transfected U87 and LN-Z308) cells similarly underwent G2-M arrest in response to TMZ, but this arrest was accompanied by only minor changes in p53 or p21(Waf1/Cip1) and was reversed within 7 days of TMZ treatment in association with the appearance of cells with either 8n or subG1 DNA content. These results suggest that glioma cells respond to TMZ by undergoing G2-M arrest. p53 is not necessary for this G2-M arrest to occur but is important in the duration of G2-M arrest and in the ultimate fate of TMZ-treated cells. Therefore, the integrity of the G2-M cell cycle checkpoint may be important in the cytotoxicity of TMZ in glioma cells.

Abrogation of the Chk1-mediated G(2) checkpoint pathway potentiates temozolomide-induced toxicity in a p53-independent manner in human glioblastoma cells.

Temozolomide (TMZ) produces O(6)-methylguanine in DNA, which in turn mispairs with thymine, triggering futile DNA mismatch repair (MMR) and ultimately cell death. We found previously that in p53-proficient human glioma cells, TMZ-induced futile DNA MMR resulted not in apoptosis but rather in prolonged, p53- and p21-associated G(2)-M arrest and senescence. Additionally, p53-deficient cells were relatively more TMZ resistant than p53-deficient glioma cells, which underwent only transient G(2)-M arrest before death by mitotic catastrophe. These results suggested that prolonged G(2)-M arrest might protect cells from TMZ-induced cytotoxicity. In the present study, we therefore focused on the mechanism by which TMZ induces G(2)-M arrest and on whether inhibition of such G(2)-M arrest might sensitize glioma cells to TMZ-induced toxicity. U87MG glioma cells treated with TMZ underwent G(2)-M arrest associated with Chk1 activation and phosphorylation of both cdc25C and cdc2. These TMZ-induced effects were inhibited by the Chk1 kinase inhibitor UCN-01. Although not in itself toxic, UCN-01 increased the cytotoxicity of TMZ 5-fold, primarily by inhibiting cellular senescence and increasing the percentage of cells bypassing G(2)-M arrest and undergoing mitotic catastrophe. In addition to enhancing TMZ-induced cytotoxicity in p53-proficient cells, UCN-01 also blocked TMZ-induced Chk1 activation and transient G(2)-M arrest in p53-deficient U87MG-E6 cells and similarly enhanced TMZ-induced mitotic catastrophe and cell death. Taken together, these results indicate that Chk1 links TMZ-induced MMR to G(2)-M arrest. Furthermore, inhibition of the cytoprotective G(2) arrest pathway sensitizes cells to TMZ-induced cytotoxicity and may represent a novel, mechanism-based means of increasing TMZ efficacy in both p53 wild-type and p53 mutant glioma cells.

Effects of streptozotocin/bis-chloroethylnitrosourea combination therapy on O6-methylguanine DNA methyltransferase activity and mRNA levels in HT-29 cells in vitro.

Treatment of chloroethylnitrosourea-resistant cells with streptozotocin (STZ) prior to bis-chloroethylnitrosourea (BCNU) exposure has been shown to result in a depletion of O6-methylguanine DNA methyltransferase (MGMT) activity, increased BCNU-induced interstrand cross-linking, and a 2-3 log enhancement of BCNU cytotoxicity in vitro. The current study was undertaken to define the kinetics of repletion of MGMT activity following the STZ/BCNU combination and to assess at the molecular level the effects of the combination on MGMT mRNA expression. Results demonstrate that MGMT activity can be depleted by greater than 90% relative to untreated controls using an optimized STZ/BCNU combination regimen and that greater than 50% depletion can be maintained for at least 24 h. This depletion appears to be independent of effects at the mRNA level because neither STZ alone nor the STZ/BCNU combination significantly altered steady state levels of MGMT mRNA. Cytotoxicity studies are consistent with MGMT repletion data and demonstrate that, as the interval between STZ and BCNU exposures increases, the degree of enhanced cytotoxicity induced by the combination relative to BCNU alone decreases. These results suggest that the enhanced cytotoxicity induced by the STZ/BCNU combination over BCNU treatment alone is favored by both the lack of induction of expression of MGMT mRNA and by slow reappearance of MGMT activity.

Formation of intracranial tumors by genetically modified human astrocytes defines four pathways critical in the development of human anaplastic astrocytoma.

The formation of human malignant gliomas is thought to involve the accumulation of multiple genetic alterations. To define the function of specific alterations in glioma formation, we serially introduced genetic alterations functionally equivalent to those noted in human malignant gliomas into normal human astrocytes (NHAs). We then monitored the ability of each of these alterations to contribute to the growth of otherwise genetically stable NHAs into intracranial malignant gliomas. Using this model, we show that expression of human telomerase catalytic component (hTERT), but not E7-mediated inactivation of pRb or E6/E7-mediated inactivation of p53/pRb, was sufficient to initiate the tumorigenic process by circumventing cellular senescence in astrocytes. hTERT expression, even in combination with inactivation of p53/pRb, did not transform astrocytes. These alterations together, however, cooperated with ras pathway activation (initiated by expression of mutant H-Ras), but not with phosphatidylinositol 3-kinase pathway activation (initiated by expression of myristoylated Akt) or epidermal growth factor receptor activation, to allow for the formation of intracranial tumors strongly resembling p53/pRb pathway-deficient, telomerase-positive, ras-activated human grade III anaplastic astrocytomas. These results identify four pathways as key in the development of human anaplastic astrocytomas.

Akt pathway activation converts anaplastic astrocytoma to glioblastoma multiforme in a human astrocyte model of glioma.

Human malignant gliomas are thought to develop as the result of stepwise accumulations of multiple genetic alterations. Recently, we showed that E6/E7-mediated inactivation of p53/pRb, ras pathway activation (initiated by expression of mutant H-Ras), and expression of human telomerase reverse transcriptase (hTERT) in combination converted normal human astrocytes into cells that formed intracranial tumors resembling human anaplastic astrocytoma (AA). In this study, we created human astrocytes that, in addition to expressing E6/E7, hTERT, and Ras, also expressed a constitutive activated form of Akt intended to mimic the Akt activation noted in grade IV glioblastoma multiforme (GBM). Although these cells grew no differently than astrocytes expressing E6, E7, and H-Ras in vitro or in the first 28 days following s.c. implantation, they ultimately formed tumors four to six times larger than those formed by the E6/E7/hTERT/Ras cells. Unlike the poorly vascularized, necrosis-free AA formed by E6/E7/hTERT/Ras cells, the tumors formed by s.c. or intracranial injection of Akt-expressing cells had large areas of necrosis surrounded by neovascularization and were consistent in appearance with grade IV human GBM. These results show that activation of the Akt pathway is sufficient to allow conversion of human AA to human GBM.

Understanding and manipulating O6-methylguanine-DNA methyltransferase expression.

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that transfers methyl and alkyl lesions from the O6 position of guanine to a cysteine in its structure. The ability of MGMT to also remove precytotoxic O6-alkylguanine lesions induced by chemotherapeutic chloroethylnitrosoureas has made down-regulation of MGMT expression the key component in strategies designed to sensitize tumors to the cytotoxic potential of chloroethylnitrosoureas. The study of how to regulate MGMT expression at the gene, mRNA, and protein levels has contributed not only to the development of effective inhibitors of MGMT action, but also, in a broader sense, to a better understanding of gene regulation and protein structure/function.

Phosphatidylserine-dependent phagocytosis of apoptotic glioma cells by normal human microglia, astrocytes, and glioma cells.

Apoptotic cells display signals that trigger phagocytic removal by macrophages or neighboring cells. To better understand the signals triggering phagocytosis of apoptotic glioma cells, and to identify the cells that might be involved in the phagocytic process, U-251 MG glioma cells were made apoptotic by etoposide (25 microg/ml) treatment and were incubated with normal human astrocytes (NHA), glioma cells, or microglia. Extent of phagocytosis was assessed by an in vitro phagocytosis assay. After 3 h of incubation with apoptotic cells, phagocytes tested were washed to remove nonengulfed cells, then fixed, stained, and counted to determine phagocytosis index (PI). NHA, glioma cells, and microglia all phagocytosed apoptotic, but not nonapoptotic, glioma cells. Microglia, however, had a PI approximately 4-fold higher than did either NHA or glioma cells. Binding of phosphatidylserine (PS) on apoptotic glioma cell membranes by annexin-V inhibited phagocytosis by 90% in both microglia and NHA. The activity of an enzyme (scramblase) that moves PS from the inner cell membrane to the outer cell membrane was also increased in apoptotic glioma cells. These results suggest that a variety of cells present in and near gliomas in vivo can remove glioma cells in a PS-dependent scramblase-mediated fashion. Manipulation of scramblase and/or PS exposure in glioma cells may therefore be a means of triggering phagocytic removal of glioma cells.

PI(3) kinase is associated with a mechanism of immunoresistance in breast and prostate cancer.

Immune escape describes a critical event whereby tumor cells adopt an immunoresistant phenotype to escape adaptive surveillance. We show that expression of a pivotal negative regulator of T-cell function, B7-H1, correlates with PI(3) kinase activation in breast and prostate cancer patients. B7-H1-mediated immunoresistance can be attenuated by inhibitors of the PI(3) kinase pathway, and is dependent on S6K1-mediated translational regulation of B7-H1 protein. Breast and prostate carcinoma cells with activated PI(3) kinase lose the immunoresistant phenotype after treatment with B7-H1 siRNA. Conversely, breast and prostate carcinoma cells with minimal PI(3) kinase activation adopt an immunoresistant phenotype when engineered to overexpress B7-H1 protein. These observations describe a mechanism for immune escape from tumor dormancy in humans that relates to oncogenesis.

DNA adenine adducts induced by nitrogen mustards and their role in transcription termination in vitro.

Previous studies have demonstrated that three cancer chemotherapeutic compounds of the nitrogen mustard class, melphalan (L-PAM), nitrogen mustard (HN2) and chlorambucil (CBC), each generated DNA lesions that prematurely terminate in vitro transcription. Sites of these lesions were inconsistent with sites of N7 guanine monoadducts formed by these compounds, and in the cases of L-PAM and CBC were suggestive of adenine lesions. The present study is an attempt to identify and characterize nitrogen mustard-induced non-N7 guanine DNA adducts, and in particular adenine DNA adducts, and to assess their role in drug-induced in vitro transcription termination. Data from studies using a modified Maxam-Gilbert DNA sequencing technique demonstrate that L-PAM and CBC, but not HN2, generate heat-labile, alkaline-stabilized adenine adducts at nearly every adenine in a region of a defined DNA template examined. Comparison of sites of L-PAM- and CBC-induced adenine adducts to known sites of drug-induced transcription termination in the same DNA template show that L-PAM- and CBC-induced transcription termination is associated not with drug lesions at single adenines, but rather with drug-induced adducts at neighboring adenines. Additional in vitro transcription studies using a small DNA molecule generated by polymerase chain reaction-mediated DNA amplification demonstrate that none of the transcription-terminating lesions induced by L-PAM and CBC in this molecule are interstrand in nature. These results suggest that some, but not all, nitrogen mustard compounds can generate heat-labile adenine lesions in DNA, and that bifunctional nitrogen mustards that can form heat-labile adenine adducts also form adducts consistent with intrastrand adenine-adenine crosslinks. These adducts at pairs of adenines in turn appear to be responsible for L-PAM- and CBC-induced transcription termination in vitro.

In vitro transcription termination by N,N'-bis(2-chloroethyl)-N-nitrosourea-induced DNA lesions.

N,N'-Bis(2-chloroethyl)-N-nitrosourea (BCNU) and its derivatives are chemotherapeutic DNA-damaging agents that generate a variety of monoadducts, intrastrand cross-links, and interstrand cross-links. The cytotoxic potential of the compounds has been linked to their ability to form DNA interstrand cross-links, which presumably inhibit subsequent DNA replication. To address the possibility that BCNU-induced lesions may also influence other DNA-directed actions such as transcription, and to identify the DNA lesions involved, a synthetic DNA template containing phage RNA polymerase promoters at both ends was incubated with BCNU and, after drug removal, transcribed in vitro. For comparison, similar studies were carried out with cis-diammine-dichloroplatinum(II) and trans-diamminedichloroplatinum(II), which are known to induce defined transcription-terminating lesions. The results suggest that BCNU, like platinum compounds, can induce lesions resulting in termination of transcription in vitro, although the predominant transcription-terminating lesions, unlike those produced by cis-diamminedichloroplatinum(II), most likely represent interstrand DNA cross-links.