Eric H. Radany

Novel mechanisms of E2F induction by BK virus large-T antigen: requirement of both the pRb-binding and the J domains.

ABSTRACT E2F activity is regulated in part by the retinoblastoma family of tumor suppressor proteins. Viral oncoproteins, such as simian virus 40 (SV40) large-T antigen (TAg), adenovirus E1A, and human papillomavirus E7, can disrupt the regulation of cellular proliferation by binding to pRb family members and dissociating E2F-pRb family protein complexes. BK virus (BKV), which infects a large percentage of the human population and has been associated with a variety of human tumors, encodes a TAg homologous to SV40 TAg. It has been shown that BKV TAg, when expressed at low levels, does not detectably bind to pRb family members, yet it induces a serum-independent phenotype and causes a decrease in the overall levels of pRb family proteins. The experiments presented in this report show that, despite the lack of TAg-pRb interactions, BKV TAg can induce transcriptionally active E2F and that this induction does in fact require an intact pRb-binding domain as well as an intact J domain. In addition, E2F-pRb family member complexes can be detected in both BKV and SV40 TAg-expressing cells. These results suggest the presence of alternate cellular mechanisms for the release of E2F in addition to the well-established model for TAg-pRb interactions. These results also emphasize a role for BKV TAg in the deregulation of cellular proliferation, which may ultimately contribute to neoplasia.

Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells

Purpose: We have reported previously that the expression of E. coli dUTPase (dutE) can protect HT29 cells from 5-fluorodeoxyuridine (FdUrd)-induced DNA fragmentation and cytotoxicity. In the study reported here, we further characterized the ability of dutE expression in one HT29 clone, dutE7, to alter the effects of treatment with FdUrd and other thymidylate synthase (TS) inhibitors. In addition, we developed two HuTu80 dutE-expressing clones using a pLNCX-dutE retroviral construct and tested their sensitivity to FdUrd-induced DNA fragmentation and cytotoxicity. Methods: Both a dutE retroviral expression system and a dutE antibody were developed to facilitate the generation and screening of dutE-expressing clones. HT29 and HuTu80 clones expressing dutE were tested for drug-induced DNA damage with either alkaline elution or pulsed field gel electrophoresis and drug-induced loss of clonogenicity. Results: Following a 24-h treatment with 100 μM CB3717 or 500 nM methotrexate (MTX), dutE7 cells were significantly less sensitive to drug-induced loss of clonogenicity than con3 cells. DutE7 cells were also resistant to CB3717-induced DNA fragmentation at 24 h. However, following a 48-h treatment with CB3717 or MTX there was no difference in survival between con3 and dutE7 cells, even though DNA damage was still greatly attenuated in the dutE7 cell line. In addition, expression of dutE in two HuTu80 clones, 80  C and 80  K, did not protect these cells from FdUrd-induced DNA damage or cytotoxicity. Conclusions: We conclude that the role of uracil misincorporation and subsequent DNA damage in cytotoxicity induced by TS inhibitors, in HT29 cells, is time dependent, and that cytotoxicity caused by long-term exposure to these drugs is largely independent of resultant DNA damage, in this cell line. The inability of dutE to protect HuTu80 cells from FdUrd further suggests that the significance of uracil misincorporation resulting from TS inhibition varies among cell lines.

A brain tumor dose escalation protocol based on effective dose equivalence to prior experience

PURPOSE The current study describes the design of a dose escalation protocol for conformal irradiation of primary brain tumors that preserves the safe experience of a previous, sequential dose escalation scheme while enabling the delivery of substantially higher effective doses to a central target volume. METHODS AND MATERIALS Normalized isoeffective composite dose distributions were formed for 20 patients treated on the original protocol (which specified three progressively smaller planning target volumes [PTVs]) using the linear quadratic model (here corrected to equivalent 2 Gy fractions using alpha/beta=10 Gy). These distributions were investigated and a new protocol was designed to preserve a similar level of efficacy and lack of toxicity for the outer volumes, but allowing a higher dose to the inner PTV. Treatment plans were then investigated to determine if the objectives of the new protocol were achievable. In particular, plans that simultaneously achieved all biological treatment planning objectives (all fields treated each day) were investigated. Finally, the success of the protocol design was demonstrated by analysis of the effective dose distributions of 10 patients treated using the new protocol. RESULTS The composite normalized isoeffective minimum doses to the outer PTVs (PTV3 and PTV2) in the original protocol were close to 60 Gy and 75 Gy, respectively, and these values are specified as the minimum doses to those volumes for the new protocol. Homogeneity requirements to maintain equivalence for the outer target volume domains are: not more than 25% of [PTV3 exclusive of PTV2] >75 Gy; and not more than 50% of [PTV2 exclusive of PTV1] >85 Gy. Treatment plans using multiple noncoplanar arrangements of beams and static intensity modulation treat all volumes at each session. DVHs of the normalized isoeffective dose distributions reveal the equivalence of the new protocol plans to the sequential plans in the previous protocol as well as the ability to achieve a higher dose of 90 Gy to the isocenter of PTV1 (+/-5% homogeneity required). CONCLUSION The ability to incorporate past experience through use of the linear quadratic model in the design of a new dose escalation protocol is demonstrated.

Lack of dependence of 5-fluorodeoxyuridine-mediated radiosensitization on cytotoxicity

It has been proposed that fluoropyrimidine-mediated cytotoxicity and radiosensitization are closely correlated. We have shown that HT29 human colon cancer cells transfected with the E. coli dUTPase gene are resistant to 5-fluorodeoxyuridine (FdUrd)-mediated cytotoxicity, presumably through more effective elimination of dUTP. We used these cells to assess the association between radiosensitization and cytotoxicity produced by FdUrd. The radiation sensitivities of the clones expressing elevated dUTPase activity (dutE clones) were similar to those of untransfected HT29 cells or HT29 cells which had been transfected with only the expression vector for the E. coli gene (con clones). We found that FdUrd produced similar increases in radiation sensitivity regardless of dUTPase activity. Levels of dUTPase in the dutE clones remained elevated during the entire period of FdUrd exposure, demonstrating that the lack of difference between dutE and Con clones was not a reflection of down-regulation of dUTPase activity by FdUrd. Flow cytometry showed that all clones progressed past the G1/S-phase boundary and into early S phase during FdUrd treatment. These data suggest that the mechanisms of FdUrd-mediated cytotoxicity and radiosensitization are not closely linked. These findings, combined with our previous investigations, are consistent with the hypothesis that radiosensitization occurs in cells which progress past the G1/S-phase boundary in the presence of FdUrd.

14 Enzymes That Incise Damaged DNA

Publisher Summary This chapter focuses on DNA-incising activities from Escherichia coli . E.coli has been the subject of extensive genetic and biochemical investigations on the excision repair of DNA. The excision of damaged or inappropriate nucleotides from DNA can occur by a number of different biochemical pathways, depending on both the nature of the specific base damage in question and on the particular organism under investigation. Most pathways of excision repair of DNA include the enzyme-catalyzed hydrolysis of phosphodiester bonds by specific enzymes. Such enzymes can be divided into two major classes (1) those that attack phosphodiester bonds in DNA subsequent to the hydrolysis of the associated glycosylic bond that links a nitrogenous base to the deoxyribose-phosphate backbone and (2) those that directly attack phosphodiester bonds in damaged DNA. The former class of enzymes is designated as apurinic/apyrimidinic (AP) endonucleases because their endonuclease activity is confined to sites of base loss in DNA. Such substrate sites arise by the spontaneous hydrolysis of N-glycosylic bonds in DNA, or by enzyme-catalyzed hydrolysis of these bonds by DNA glycosylases.

Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway: II: host constitutive expression, repair DNA synthesis, and in vitro activity

In the Bacillus subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, sometimes followed by productive cell infection. Previous studies have shown that ultraviolet (UV)-irradiation of the competent host cells, or cotransfection of UV-irradiated heterologous DNA, can increase the efficiency of transfection in some cases; these latter two phenomena have been called transfection enhancement (TE). In an accompanying paper, we show that TE is apparently confined to the B. subtilis phages that contain hydroxymethyluracil (HMU) in their DNA, and that the photoproduct in UV-irradiated DNA that mediates TE is specific, and different than the pyrimidine dimer, thymine glycol, uracil, or HMU. We also show that TE is due to reduced intracellular endonucleolytic attack of transfecting DNA. Based on this DNA base and nucleolytic specificity, we hypothesized that TE reflects the incidental action of a host DNA repair system on transfecting HMU phage DNA. In continuing these studies, we show here that duplex infecting HMU phage DNA is apparently inactivated by this same putative repair system when phage protein synthesis is blocked. We find, too, that this inactivation of infecting HMU phage DNA can be inhibited by UV-irradiated DNA, and that this process has a similar DNA base specificity as for TE. The survival of infecting HMU phage DNA is dependent on host DNA polymerase activity. We can detect specific DNA synthesis consistent with formation of repair patches when inactivation of infecting HMU phage DNA is ongoing, but not when it is inhibited by the presence of UV DNA or by allowing phage gene expression. Each of these results is consistent with the hypothesis that TE reflects the action of a novel DNA repair pathway. We show that a candidate TE-associated enzymatic activity can be detected in cell free extracts of uninfected, but not HMU phage-infected, B. subtilis cells. Correspondingly, the extracts of phage-infected cells appear to contain a diffusible factor that acts as an inhibitor of this host enzyme.

Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway: I: DNA base and nucleolytic specificity

Cells of Bacillus subtilis can enter a natural physiological state, termed competence, that is permissive for uptake of DNA from the surrounding medium. In the B. subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, followed by intracellular processing that may ultimately lead to productive infection. Previous investigations have shown that transfecting DNA is usually far less infectious (on a molar basis) than is the DNA injected by phage particles; this result is apparently due to inactivating events suffered by transfecting DNA during its metabolism by competent cells. Earlier studies also demonstrated that, in some cases, the infectivity of transfecting DNA can be increased by ultraviolet (UV) irradiation of the competent cells prior to transfection, or by cotransfection of UV-irradiated heterologous DNAs; collectively, these phenomena have been termed transfection enhancement (TE). We propose here that some transfecting B. subtilis phage DNAs are attacked by a novel host DNA repair system, and that TE reflects inhibition of this by a competing substrate in UV-irradiated DNA. In support of this model, we show that UV-DNA cotransfection leads to a reduced rate of intracellular endonucleolytic breakdown of transfecting DNA. We also demonstrate that TE displays marked specificity of a kind frequently observed for repair enzymes. Thus, phages that contain hydroxymethyl uracil (HMU), but not thymine, in their genomes are susceptible to this process. In addition, we show that the photoproduct(s) in UV-irradiated DNA that produces TE by cotransfection is specific, and is not uracil, a pyrimidine dimer, thymine glycol, HMU, or a substrate for the E. coli thymine glycol DNA N-glycosylase. This photoproduct is derivable from thymine or HMU. The implications of these results are discussed.

Effect of bromodeoxyuridine on radiation-induced dna damage based on DNA fragment size using pulsed field gel electrophoresis

We have used biphasic linear ramping pulsed-field gel electrophoresis (PFGE) to understand the effect of incorporation of bromodeoxyuridine (BrdUrd) on radiation-induced DNA damage and repair. This technique permits a determination of the fragment size distribution produced immediately after irradiation as well as during the repair period. We found that incorporation of BrdUrd increased the induction and decreased the repair of radiation damage. The fragment size distribution was consistent with a random breakage model. When we found that significantly more damage was detected after irradiation of deproteinized DNA compared to intact cells, we studied the effects of BrdUrd incorporation on the radiation response of cells or DNA at various phases of preparation for electrophoresis: cells adherent to the culture dish (A), trypsinized cells (B), agarose-embedded cells (C) and deproteinized DNA (D). Although there was a general tendency to detect more damage when irradiation was performed later in the prepara...