Antitumor effect of therapeutic HPV DNA vaccines with chitosan-based nanodelivery systems

Background

Cervical cancer is the second-most-common cause of malignancies in women worldwide, and the oncogenic activity of the human papilloma virus types (HPV) E7 protein has a crucial role in anogenital tumors. In this study, we have designed a therapeutic vaccine based on chitosan nanodelivery systems to deliver HPV-16 E7 DNA vaccine, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer. We have developed a Nano-chitosan (NCS) as a carrier system for intramuscular administration using a recombinant DNA vaccine expressing HPV-16 E7 (NCS-DNA E7 vaccine). NCS were characterized in vitro for their gene transfection ability.

Results

The transfection of CS-pEGFP NPs was efficient in CHO cells and the expression of green fluorescent proteins was well observed. In addition, NCS-DNA E7 vaccine induced the strongest E7-specific CD8+ T cell and interferon γ responses in C57BL/6 mice. Mice vaccinated with NCS-DNA E7 vaccine were able to generate potent protective and therapeutic antitumor effects against challenge with E7-expressing tumor cell line, TC-1.

Conclusions

The strong therapeutic effect induced by the Chitosan-based nanodelivery suggest that nanoparticles may be an efficient carrier to improve the immunogenicity of DNA vaccination upon intramuscular administration and the platform could be further exploited as a potential cancer vaccine candidate in humans.     

Participation of the host protein(s) in the morphogenesis of bacteriophage P22

Summary Spontaneous mutants of S. typhimurium resistant to thiolutin are conditionally non-permissive for phage P22 development (Joshi and Chakravorty 1979). At 40° C non-infective phage particles are produced. Phage development in two nonpermissive hosts (18/MC4 and 153/MC4) has been studied in detail. The steps at which the phage morphogenesis is interfered with differ in the two mutants. The electron micrograph of the particles produced in the mutant 18/MC4 reveals the presence of normal-looking particles; these particles contain phage DNA, adsorb to the permissive host but fail to inject their DNA. The particles produced in the mutant 153/MC4 which fail to adsorb to the host are found to be tail fibre-less. These observations indicate the involvement of host protein(s) in phage P22 morphogenesis.     

Removing residual DNA from Vero-cell culture-derived human rabies vaccine by using nuclease

The clearance of host cell DNA is a critical indicator for Vero-cell culture-derived rabies vaccine. In this study, we evaluated the clearance of DNA in Vero-cell culture-derived rabies vaccine by purification process utilizing ultrafiltration, nuclease digestion, and gel filtration chromatography. The results showed that the bioprocess of using nuclease decreased residual DNA. Dot-blot hybridization analysis showed that the residual host cell DNA was <100 pg/ml in the final product. The residual nuclease in rabies vaccine was less than 0.1 ng/ml protein. The residual nuclease could not paly the biologically active role of digestion of DNA. Experiments of stability showed that the freeze-drying rabies virus vaccine was stable and titers were >5.0 IU/ml. Immunogenicity test and protection experiments indicated mice were greatly induced generation of neutralizing antibodies and invoked protective effects immunized with intraperitoneal injections of the rabies vaccine. These results demonstrated that the residual DNA was removed from virus particles and nuclease was removed by gel filtration chromatography. The date indicated that technology was an efficient method to produce rabies vaccine for human use by using nuclease.     

Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass (Lates calcarifer) to protect from Vibrio (Listonella) anguillarum

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass (Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.     

Germinal proto-mitochondria from rat liver

A number of properties of the smallest (less than 0.2 μm) germinal proto-mitochondria (PRMC) from rat liver have been studied. These PRMC were obtained by filtering the light fraction of hepatic mitochondria (MC) through calibrated millipore membranes. Germinal PRMC contain in general the same proteins as MC. However, they have the reduced content of flavoproteins and zero cytochrome oxidase. Germinal PRMC, in contrast to MC, almost does not contain the “aging pigment” - lipofuscin. They have DNA; the DNA/protein ratio in them is much higher than in MC, i.e. they are poor in protein. The obtained results support the earlier assumption that MC in specialized animal cells can arise from germinal PRMC - particles smaller than 0.2 μm containing DNA. It is assumed that the DNA molecules enter to cytoplasm during degradation of old MC serves as a seed for the formation of PRMC (with the connection of nuclear DNA).     

钠米颗粒介导质粒DNA转染体外真核细胞

DNA传递是基因表达与功能研究及其医学应用的重要技术,安全高效的DNA传递一直是研究者期待的目标。利用一种新的阳离子多聚物脱乙酰甲壳胺16介导重组质粒pcDNA3vp1转染COS7细胞,RTPCR可检测到目的基因vp1在mRNA水平的表达,实时定量PCR结果表明其转染效率介于脂质体与磷酸钙法之间,同时还对转染条件进行了探讨。DNA结合分析发现脱乙酰甲壳胺16能够与DNA形成核酸纳米颗粒,提高DNA稳定性,促进真核细胞转染效率的提高。这些结果表明脱乙酰甲壳胺16确能做为一种新型的非病毒纳米DNA传递载体,并将可能在基因表达与功能研究及基因治疗等领域发挥重要作用 。     

Xenogeneic Human p53 DNA Vaccination by Electroporation Breaks Immune Tolerance to Control Murine Tumors Expressing Mouse p53

The pivotal role of p53 as a tumor suppressor protein is illustrated by the fact that this protein is found mutated in more than 50% of human cancers. In most cases, mutations in p53 greatly increase the otherwise short half-life of this protein in normal tissue and cause it to accumulate in the cytoplasm of tumors. The overexpression of mutated p53 in tumor cells makes p53 a potentially desirable target for the development of cancer immunotherapy. However, p53 protein represents an endogenous tumor-associated antigen (TAA). Immunization against a self-antigen is challenging because an antigen-specific immune response likely generates only low affinity antigen-specific CD8⁺ T-cells. This represents a bottleneck of tumor immunotherapy when targeting endogenous TAAs expressed by tumors. The objective of the current study is to develop a safe cancer immunotherapy using a naked DNA vaccine. The vaccine employs a xenogeneic p53 gene to break immune tolerance resulting in a potent therapeutic antitumor effect against tumors expressing mutated p53. Our study assessed the therapeutic antitumor effect after immunization with DNA encoding human p53 (hp53) or mouse p53 (mp53). Mice immunized with xenogeneic full length hp53 DNA plasmid intramuscularly followed by electroporation were protected against challenge with murine colon cancer MC38 while those immunized with mp53 DNA were not. In a therapeutic model, established MC38 tumors were also well controlled by treatment with hp53 DNA therapy in tumor bearing mice compared to mp53 DNA. Mice vaccinated with hp53 DNA plasmid also exhibited an increase in mp53-specific CD8⁺ T-cell precursors compared to vaccination with mp53 DNA. Antibody depletion experiments also demonstrated that CD8⁺ T-cells play crucial roles in the antitumor effects. This study showed intramuscular vaccination with xenogeneic p53 DNA vaccine followed by electroporation is capable of inducing potent antitumor effects against tumors expressing mutated p53 through CD8⁺ T cells.     

Accelerated immune response to DNA vaccines

DNA vaccines offer considerable promise for improvement over conventional vaccines. For the crucial step of delivering DNA vaccines intracellularly, electroporation (EP) has proven to be highly effective. This method has yielded powerful humoral and cellular responses in various species, including nonhuman primates. In an attempt to further improve DNA vaccination we used micron-size gold particles (which do not bind or adsorb DNA) as a particulate adjuvant which was coinjected with DNA intramuscularly into mice, followed by EP of the target site. The presence of gold particles accelerated the antibody response significantly. Maximum titers against hepatitis B surface antigen (HBsAg) were reached after one boost in 6 weeks, whereas 8 weeks were required without particles. These immunizations were effective in protecting mice against tumor challenge with cancer cells expressing HBsAg as a surrogate cancer antigen. Computer modeling of electric fields and gene expression studies indicate that gold particles do not stimulate EP and subsequent antigen expression. The particles may act as an attractant for immune cells, especially antigen presenting cells. We conclude that particulate adjuvants combined with DNA vaccine delivery by EP reduces the immune response time and may increase vaccine efficacy. This method may become valuable for developing prophylactic as well as therapeutic vaccines. The rapid response may be of particular interest in countering bio-terrorism.     

Protective Immunity Induced by Oral Immunization with a Rotavirus DNA Vaccine Encapsulated in Microparticles

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.     

A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T cell-mediated protective immunity against colon cancer in carcinoembryonic antigen-transgenic mice

A carcinoembryonic Ag (CEA)-based DNA vaccine encoding both CEA and CD40 ligand trimer achieved effective tumor-protective immunity against murine colon carcinoma in CEA-transgenic mice by activating both naive T cells and dendritic cells. Peripheral T cell tolerance to CEA was broken in a prophylactic model by this novel, dual-function DNA vaccine, whose efficacy was further enhanced by boosts with a recombinant Ab-IL-2 fusion protein (huKS1/4-IL-2). These conclusions are supported by four lines of evidence. First, a lethal challenge of MC38-CEA-KS Ag murine colon carcinoma cells was for the first time completely rejected in 100% of experimental animals treated by oral gavage of this DNA vaccine carried by attenuated Salmonella typhimurium, followed by five boosts with huKS1/4-IL-2. Second, specific activation of dendritic cells was indicated by their marked up-regulation in expression of costimulatory molecules B7.1 (CD80), B7.2 (CD86), and ICAM-1. Third, a decisive increase over control values was observed in both MHC class I Ag-restricted cytotoxicity of CTLs from successfully vaccinated mice and secretion of proinflammatory cytokines IFN-gamma and IL-12. Fourth, activation of CTLs was augmented, as indicated by up-regulation of activity markers LFA-1, CD25, CD28, and CD69. Taken together, these results suggest that a dual-function DNA vaccine encoding CEA and CD40 ligand trimer combined with tumor-targeted IL-2 may be a promising strategy for the rational development of DNA-based cancer vaccines for future clinical applications.     

Mitomycin C linked to DNA minor groove binding agents: synthesis, reductive activation, DNA binding and cross-linking properties and in vitro antitumor activity

Mitomycin C (MC) is a natural cytotoxic agent used in clinical anticancer chemotherapy. Its antitumor target appears to be DNA. Upon bioreductive activation MC alkylates and cross-links DNA. MC derivatives were synthesized in which MC was linked to DNA minor groove binding agents, analogous to netropsin and distamycin. One, two and three N-methylpyrrole carboxamide units were conjugated with MC by a (CH2)5-tether to the 7-amino group of MC (11, 12 and 13, respectively). In contrast to MC 11, 12 and 13 displayed non-covalent affinity to DNA. Their bioreductive activation by NADPH-cytochrome c reductase proceeded as fast as that of MC. Metabolites arising from reductive and low-pH activation were characterized and found to be analogous to those of MC. DNA cross-linking activities were weak and decreased with an increasing number of N-methylpyrrole carboxamide units linked with the mitomycin molecule. No adducts were formed with calf thymus DNA in detectable amounts. In vitro antitumor activities of 11-13 were determined using the NCI in vitro antitumor screen. The conjugates 11-13 are growth inhibitory; however, their activities are 1.5-2 orders of magnitude lower than that of MC. COMPARE analysis indicates that the mechanism of the action of 11 and 12 correlates moderately with MC but negatively with distamycin. Conjugate 13 correlates neither with MC nor with distamycin. The results suggest that the basic cause of the observed low activity of the MC-minor groove binder conjugates is the fast irreversible decay of the activated MC, competing effectively with the slow drug delivery to CpG sites, required for the alkylation.     

Interactions of surface-confined DNA with electroreduced mitomycin C comparison with acid-activated mitomycin C

The anticancer activity of the antineoplastic drug mitomycin C (MC) was investigated using transfer stripping cyclic voltammetry (TSCV) with single-stranded DNA-modified hanging mercury drop electrode (HMDE). Reductive activation of MC is necessary for drug covalent binding to DNA, and we have found that some potential-controlled interactions of MC with DNA occur at the electrode, i.e. MC can be activated by electroreduction. Acid and electroreductive MC activations were compared and different adducts were subsequently generated, suggesting that the drug can bind to DNA in more than one way. Under conditions of acid activated MC, a monofunctional adduct between C-1 of MC and N-7 of guanine was formed on the electrode surface, reduced at - 0.44 V (vs. SCE). However, when the DNA-modified electrode was immersed in a MC solution and potentials corresponding to the quinone moiety reduction (- 0.3 V or more negative vs. SCE) were applied, an intrastrand bifunctional adduct between C-1 and C-10 of MC and two N-7 of a pair of adjacent guanines in ssDNA were formed at the electrode, reduced at - 0.49 V, i.e. 50 mV more negative than the monoadduct. The results presented in this paper show for the first time electrochemical detection of DNA-MC adducts at the hanging mercury drop electrode.     

Papillomavirus pseudovirus: a novel vaccine to induce mucosal and systemic cytotoxic T-lymphocyte responses

Intestinal mucosa is a portal for many infectious pathogens. Systemic immunization, in general, does not induce a cytotoxic T-lymphocyte (CTL) response at the mucosal surface. Because papillomavirus (PV) naturally infects mucosa and skin, we determined whether PV pseudovirus, i.e., PV-like particles in which unrelated DNA plasmids are packaged, could generate specific mucosal immunity. We found that the pseudovirus that encoded the lymphocytic choriomeningitis virus gp33 epitope induced a stronger CTL response than a DNA vaccine (plasmid) encoding the same epitope given systemically. The virus-like particles that were used to make the pseudoviruses provided an adjuvant effect for induction of CTLs by the DNA vaccine. The PV pseudovirus pseudoinfected mucosal and systemic lymphoid tissues when administered orally. Oral immunization with the pseudovirus encoding human PV type 16 mutant E7 induced mucosal and systemic CTL responses. In comparison, a DNA vaccine encoding E7, when given orally, did not induce a CTL response in intestinal mucosal lymphoid tissue. Further, oral immunization with the human PV pseudovirus encoding E7 protected mice against mucosal challenge with an E7-expressing bovine PV pseudovirus. Thus, PV pseudovirus can be used as a novel vaccine to induce mucosal and systemic CTL responses.     

Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine.

We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.     

噬菌体呈现技术制备结肠癌抗独特型抗体

 以纯化的鼠抗人结肠癌细胞单克隆抗体 (简称单抗 )MC5与钥孔血蓝素 (KLH)的交联物经腹腔免疫Balb c小鼠 ,取脾分离mRNA .RT PCR分别扩增抗体重、轻链可变区基因片段 (VH 和VLcD NA ,大小分别约为 340bp和 32 0bp) ,二者经linkerDNA连接形成ScFv(singlechainvariablefragment)DNA(约 75 0bp) .将ScFvDNA与噬菌粒载体pCANTAB5E的连接产物转化于大肠杆菌TG1,经辅助噬菌体M13KO7感染后 ,获得重组噬菌体抗体ScFv文库 .以单抗MC5对ScFv文库进行 4轮亲和筛选后 ,随机挑取 80个克隆经酶联免疫吸附实验 (ELISA)筛选出 2 2个呈现ScFv形式抗独特型抗体(抗 IdScFv)的噬菌体单克隆 .竞争抑制实验表明 ,在 2 2个阳性克隆中有 4个克隆所呈现的抗 IdScFv属β或γ型 .针对单抗MC5的噬菌体呈现型抗 IdScFv的制备 ,为筛选新的结肠癌重组抗 Id瘤苗候选分子奠定了基础     

Simple, fast and accurate implementation of the diffusion approximation algorithm for stochastic ion channels with multiple states

BACKGROUND: The phenomena that emerge from the interaction of the stochastic opening and closing of ion channels (channel noise) with the non-linear neural dynamics are essential to our understanding of the operation of the nervous system. The effects that channel noise can have on neural dynamics are generally studied using numerical simulations of stochastic models. Algorithms based on discrete Markov Chains (MC) seem to be the most reliable and trustworthy, but even optimized algorithms come with a non-negligible computational cost. Diffusion Approximation (DA) methods use Stochastic Differential Equations (SDE) to approximate the behavior of a number of MCs, considerably speeding up simulation times. However, model comparisons have suggested that DA methods did not lead to the same results as in MC modeling in terms of channel noise statistics and effects on excitability. Recently, it was shown that the difference arose because MCs were modeled with coupled gating particles, while the DA was modeled using uncoupled gating particles. Implementations of DA with coupled particles, in the context of a specific kinetic scheme, yielded similar results to MC. However, it remained unclear how to generalize these implementations to different kinetic schemes, or whether they were faster than MC algorithms. Additionally, a steady state approximation was used for the stochastic terms, which, as we show here, can introduce significant inaccuracies. MAIN CONTRIBUTIONS: We derived the SDE explicitly for any given ion channel kinetic scheme. The resulting generic equations were surprisingly simple and interpretable--allowing an easy, transparent and efficient DA implementation, avoiding unnecessary approximations. The algorithm was tested in a voltage clamp simulation and in two different current clamp simulations, yielding the same results as MC modeling. Also, the simulation efficiency of this DA method demonstrated considerable superiority over MC methods, except when short time steps or low channel numbers were used.     

Characterization of hepatitis B virus DNA polymerase

Hepatitis B virus (HBV) particles were separated from the blood-plasma containing HBe and HBs antigens (subtype adr) and the nature of the endogenous DNA polymerase in the HBV core particles was studied. The HBV endogenous DNA polymerase activity was examined under the conditions used for preparation of HBV vaccine. The endogenous DNA polymerase activity was reduced slowly upon the heat treatment or the formalin treatment. The reductions of the activity were 65% and 70% upon the heat treatment at 60 C for 10 hr and the formalin treatment at 37 C for 90 hr, respectively. Properties of the HBV endogenous DNA polymerase were studied by utilizing specific inhibitors against the eukaryotic DNA polymerases. Our results showed that the HBV endogenous DNA polymerase is resistant to aphidicolin and N-ethylmaleimide, and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, phosphonoformic acid and 9-beta-D-arabinofuranosyladenosine 5'-triphosphate.     

The effects of DNA formulation and administration route on cancer therapeutic efficacy with xenogenic EGFR DNA vaccine in a lung cancer animal model

Background

Tyrosine kinase inhibitor gefitinib is effective against lung cancer cells carrying mutant epidermal growth factor receptor (EGFR); however, it is not effective against lung cancer carrying normal EGFR. The breaking of immune tolerance against self epidermal growth factor receptor with active immunization may be a useful approach for the treatment of EGFR-positive lung tumors. Xenogeneic EGFR gene was demonstrated to induce antigen-specific immune response against EGFR-expressing tumor with intramuscular administration.

Methods

In order to enhance the therapeutic effect of xenogeneic EGFR DNA vaccine, the efficacy of altering routes of administration and formulation of plasmid DNA was evaluated on the mouse lung tumor (LL2) naturally overexpressing endogenous EGFR in C57B6 mice. Three different combination forms were studied, including (1) intramuscular administration of non-coating DNA vaccine, (2) gene gun administration of DNA vaccine coated on gold particles, and (3) gene gun administration of non-coating DNA vaccine. LL2-tumor bearing C57B6 mice were immunized four times at weekly intervals with EGFR DNA vaccine.

Results

The results indicated that gene gun administration of non-coating xenogenic EGFR DNA vaccine generated the strongest cytotoxicty T lymphocyte activity and best antitumor effects. CD8(+) T cells were essential for anti-tumor immunityas indicated by depletion of lymphocytes in vivo.

Conclusion

Thus, our data demonstrate that administration of non-coating xenogenic EGFR DNA vaccine by gene gun may be the preferred method for treating EGFR-positive lung tumor in the future.     

血源乙肝疫苗的电镜制样方法

用作样品支持物喷碳的铜网,磷钨酸染色制备血源乙肝疫苗电镜检测样品。４９３批血源乙肝疫苗的镜检结果显示：经溴化钾和蔗糖离心后,原料血浆中的Ｄａｎｅ颗粒和管形颗粒已去除,只观察到２２－２４纳米的ＨＢｓＡｇ球形颗粒。     

Synthesis of Mitomycin C and Decarbamoylmitomycin C N2 deoxyguanosine-adducts

Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) – a derivative of MC lacking the carbamate on C10 – are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA monoadducts as well as intrastrand and interstrand cross-links (ICLs). The major monoadducts generated by MC and DMC in tumor cells have opposite stereochemistry at carbon one of the guanine–mitosene bond: trans (or alpha) for MC and cis (or beta) for DMC. We hypothesize that local disruptions of DNA structure from trans or cis adducts are responsible for the different biochemical responses produced by MC and DMC. Access to DNA substrates bearing cis and trans MC/DMC lesions is essential to verify this hypothesis. Synthetic oligonucleotides bearing trans lesions can be obtained by bio-mimetic methods. However, this approach does not yield cis adducts. This report presents the first chemical synthesis of a cis mitosene DNA adduct. We also examined the stereopreference exhibited by the two drugs at the mononucleotide level by analyzing the formation of cis and trans adducts in the reaction of deoxyguanosine with MC or DMC using a variety of activation conditions. In addition, we performed Density Functional Theory calculations to evaluate the energies of these reactions. Direct alkylation under autocatalytic or bifunctional conditions yielded preferentially alpha adducts with both MC and DMC. DFT calculations showed that under bifunctional activation, the thermodynamically favored adducts are alpha, trans, for MC and beta, cis, for DMC. This suggests that the duplex DNA structure may stabilize/oriente the activated pro-drugs so that, with DMC, formation of the thermodynamically favored beta products are possible in a cellular environment.