Determining Duration of HER2-Targeted Therapy Using Stem Cell Extinction Models

Introduction

Trastuzumab dramatically improves survival in breast cancer patients whose tumor overexpresses HER2. A subpopulation of cells in human breast tumors has been identified with characteristics of cancer stem cells. These breast cancer stem-like cells (BCSCs) rely on HER2 signaling for self-renewal, suggesting that HER2-targeted therapy targets BCSCs even when the bulk of the tumor does not overexpress HER2. In order to guide clinical trials examining HER2-targeted therapy in the adjuvant setting, we propose a mathematical model to examine BCSC population dynamics and predict optimal duration of therapy.

Methods

Varying the susceptibility of BCSCs to HER2-targeted therapy, we quantify the average time to extinction of BCSCs. We expand our model using stochastic simulation to include the partially differentiated tumor cells (TCs) that represent bulk tumor population and examine effects of plasticity on required duration of therapy.

Results

Lower susceptibility of BCSCs and increased rates of dedifferentiation entail longer extinction times, indicating a need for prolonged administration of HER2-targeted therapy. We predict that even when therapy does not appreciably reduce tumor size in the advanced cancer setting, it will eventually eradicate the tumor in the adjuvant setting as long as there is at least a modest effect on BCSCs.

Conclusions

We anticipate that our results will inform clinical trials of targeted therapies in planning the duration of therapy needed to eradicate BCSCs. Our predictions also address safety, as longer duration of therapy entails a greater potential impact on normal stem cells that may also be susceptible to stem cell-targeted therapies.     

Investigation of the survival viability of cervical cancer cells (HeLa) under visible light induced photo-catalysis with facile synthesized WO3/ZnO nanocomposite

The photo catalytic degradation, a proven chemical process used for the decontamination of organic/inorganic pollutants and microorganisms in water was implemented. In this work for the selective killing of cervical cancer cells (HeLa cells) by using nano-composite of ZnO (Zinc Oxcide), WO₃ (tungsten oxide) and (n-WO₃/ZnO) as a photo-catalyst under the irradiation of visible light. All the three nanostructured semiconducting materials (WO3, ZnO and n-WO3/ZnO) were synthesized by facile chemical precipitation method and their morphological and optical characterization studies were carried out to elucidate the observed enhancement in the photo-catalytic killing of HeLa cancer cells with n-WO3/ZnO as a photo-catalyst. After 60 min of photo-catalytic reaction with n-WO₃/ZnO as a photo-catalyst, a survival viability of HeLa cancer cells as low as 15% was achieved (nearly 85% of killing), as compared to 65% of HeLa cancer cell survival viability (nearly 35% of killing) with individual use of WO₃ and ZnO as photo-catalysts under the same irradiation and experimental conditions. This improved photo-catalytic killing of HeLa cancer cells using n-WO₃/ZnO in the visible spectral region is attributed to the enhanced visible light absorption and reduced electron hole recombination, characteristically brought about in the n-WO₃/ZnO composite material. As photo-catalytic killing of the cancer cells can be selective, localized and reasonably efficient, in principle, this method can be considered as a non-invasive targeted treatment option for killing any type of cancer cells. HeLa cells, in particular are the cervical cancer cell and the tumors in and around cervix, containing HeLa cells can be non-surgically accessed and photo-catalytically treated with appropriate photo-catalyst and light source.     

A changed nitrogenase activity in Rhodospirillum rubrum after substitution of tungsten for molybdenum

The nitrogenase activity in Rhodospirillum rubrum was changed when the cells were made either Mo-deficient or when Mo was replaced by tungsten (W) as trace element in the growth medium: In the absence of N₂, normal Mo cells evolved H₂ (via nitrogenase) from added malate in the light faster than W cells, which in turn evolved H₂ faster than Mo-deficient cells. In the presence of N₂, on the other hand, nitrogen fixation rate in W cells was very close to the low rate found with Mo-deficient cells. Incubation after harvesting of Mo-deficient cells with 2×10^-5 M molybdate or tungstate stimulated the H₂ evolution (similarly with both trace elements) as well as the N₂ fixation (Mo was more effective than W). This indicates that the nitrogenase activity of W cells was truly caused by W and not merely by remaining traces of Mo. The ATP consumption is apparently higher with a W-containing nitrogenase than with the normal Mo-nitrogenase. Further, the affinity to N₂ of the W cells seems to be lower than with the Mo cells.     

Synthesis and anti-proliferative activity of aromatic substituted 5-((1-benzyl-1H-indol-3-yl)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione analogs against human tumor cell lines

Based on previous SAR studies on N-benzylindole and barbituric acid hybrid molecules, we have synthesized a series of aromatic substituted 5-((1-benzyl-1H-indol-3-yl)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione analogs (3a–i) and evaluated them for their in vitro growth inhibition and cytotoxicity against a panel of 60 human tumor cell lines. Compounds 3c, 3d, 3f and 3g were identified as highly potent anti-proliferative compounds against ovarian, renal and breast cancer cell lines with GI₅₀ values in low the nanomolar range. The 4-methoxy-N-benzyl analog (3d) was the most active compound with GI₅₀ values of 20 nM and 40 nM against OVCAR-5 ovarian cancer cells and MDA-MB-468 breast cancer cells, respectively. Two other analogs, 3c (the 4-methyl-N-benzyl analog) and 3g (the 4-fluoro-N-benzyl analog) exhibited equimolar potency against MDA-MB-468 cells GI₅₀ = 30 nM). Analog 3f (the 4-chloro-N-benzyl analog) exhibited a GI₅₀ value of 40 nM against renal cancer cell line A498. These results suggest that aromatic substituted N-benzylindole dimethylbarbituric acid hybrids may have potential for development as clinical candidates to treat a variety of solid tumors.     

Discovery of N-aryl-9-oxo-9H-fluorene-1-carboxamides as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 1. Structure–activity relationships of the carboxamide group

N-(2-Methylphenyl)-9-oxo-9H-fluorene-1-carboxamide (2a) was identified as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 2a was found to be active with sub-micromolar potencies for both caspase induction and growth inhibition in T47D human breast cancer, HCT116 human colon cancer, and SNU398 hepatocellular carcinoma cancer cells. It arrested HCT116 cells in G₂/M followed by apoptosis as assayed by the flow cytometry. Structure–activity relationship (SAR) studies of the carboxamide group identified the lead compound N-(2-(1H-pyrazol-1-yl)phenyl)-9-oxo-9H-fluorene-1-carboxamide (6s). Compound 6s, with increased aqueous solubility, was found to retain the broad activity in the caspase activation assay and in the cell growth inhibition assay with sub-micromolar EC₅₀ and GI₅₀ values in T47D, HCT116, and SNU398 cells, respectively.     

c-Myc Is Required for Maintenance of Glioma Cancer Stem Cells

Background

Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells.

Methodology/Principal Findings

Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G₀/G₁ phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice.

Conclusions/Significance

These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.     

Exposure of Phytopathogenic Xanthomonas spp. to Lethal Concentrations of Multiple Oxidants Affects Bacterial Survival in a Complex Manner

During plant-microbe interactions and in the environment, Xanthomonas campestris pv. phaseoli is likely to be exposed to high concentrations of multiple oxidants. Here, we show that simultaneous exposures of the bacteria to multiple oxidants affects cell survival in a complex manner. A superoxide generator (menadione) enhanced the lethal effect of an organic peroxide (tert-butyl hydroperoxide) by 1,000-fold; conversely, treatment of cells with menadione plus H₂O₂ resulted in 100-fold protection compared to that for cells treated with the individual oxidants. Treatment of X. campestris with a combination of H₂O₂ and tert-butyl hydroperoxide elicited no additive or protective effect. High levels of catalase alone are sufficient to protect cells against the lethal effect of menadione plus H₂O₂ and tert-butyl hydroperoxide plus H₂O₂. These data suggest that H₂O₂ is the lethal agent responsible for killing the bacteria as a result of these treatments. However, increased expression of individual genes for peroxide (alkyl hydroperoxide reductase, catalase)- and superoxide (superoxide dismutase)-scavenging enzymes or concerted induction of oxidative stress-protective genes by menadione gave no protection against killing by a combination of menadione plus tert-butyl hydroperoxide. However, X. campestris cells in the stationary phase and a spontaneous H₂O₂-resistant mutant (X. campestris pv. phaseoli HR) were more resistant to killing by menadione plus tert-butyl hydroperoxide. These findings give new insight into oxidant killing of Xanthomonas spp. that could be generally applied to other bacteria.     

ΔNp63α promotes cellular quiescence via induction and activation of Notch3

Genetic analysis of TP63 indicates that ΔNp63 isoforms are required for preservation of self-renewing capacity in the stem cell compartments of diverse epithelial structures; however, the underlying cellular and molecular mechanisms remain incompletely defined. Cellular quiescence is a common feature of adult stem cells that may account for their ability to retain long-term replicative capacity while simultaneously limiting cellular division. Similarly, quiescence within tumor stem cell populations may represent a mechanism by which these populations evade cytotoxic therapy and initiate tumor recurrence. Here, we present evidence that ΔNp63α, the predominant TP63 isoform in the regenerative compartment of diverse epithelial structuresm, promotes cellular quiescence via activation of Notch signaling. In HC11 cells, ectopic ΔNp63α mediates a proliferative arrest in the 2N state coincident with reduced RNA synthesis characteristic of cellular quiescence. Additionally, ΔNp63α and other quiescence-inducing stimuli enhanced expression of Notch3 in HC11s and breast cancer cell lines, and ectopic expression of the Notch3 intracellular domain (N3^ICD) was sufficient to cause accumulation in G₀/G₁ and increased expression of two genes associated with quiescence, Hes1 and Mxi1. Pharmacologic inhibition of Notch signaling or shRNA-mediated suppression of Notch3 were sufficient to bypass quiescence induced by ΔNp63α and other quiescence-inducing stimuli. These studies identify a novel mechanism by which ΔNp63α preserves long-term replicative capacity by promoting cellular quiescence and identify the Notch signaling pathway as a mediator of multiple quiescence-inducing stimuli, including ΔNp63α expression.     

Effects of N-acetylcystein on bleomycin-induced apoptosis in malignant testicular germ cell tumors

Oxidative stress has been shown to induce apoptosis in cancer cells. Therefore, one might suspect that antioxidants may inhibit reactive oxygen species (ROS) and prevent apoptosis of cancer cells. No study has been carried out so far to elucidate the effects of N-acetylcysteine (NAC) on bleomycin-induced apoptosis in human testicular cancer (NCCIT) cells. We investigated the molecular mechanisms of apoptosis induced by bleomycin and the effect of NAC in NCCIT cells. We compared the effects of bleomycin on apoptosis with H₂O₂ which directly produces ROS. Strong antioxidant NAC was evaluated alone and in combination with bleomycin or H₂O₂ in germ cell tumor-derived NCCIT cell line (embryonal carcinoma, being the nonseminomatous stem cell component). We determined the cytotoxic effect of bleomycin and H₂O₂ on NCCIT cells and measured apoptosis markers such as caspase-3, caspase-8, and caspase-9 activities and Bcl-2, Bax, and cytochrome c (Cyt-c) levels in NCCIT cells incubated with bleomycin, H₂O₂, and/or NAC. We found half of the lethal dose (LD₅₀) of bleomycin on NCCIT cell viability as 120???g/ml after incubation for 72?h. Incubation with bleomycin (LD₅₀) induced increases in caspase-3, caspase-8, and caspase-9 activities and Cyt-c and Bax protein levels and a decrease in Bcl-2 level. Co-incubation of NCCIT cells with bleomycin and 10?mM NAC abolished bleomycin-induced increases in caspase-3 and caspase-9 activities, Bax, and Cyt-c levels and bleomycin-induced decrease in Bcl-2 level. Our results indicate that bleomycin induces apoptosis in NICCT cells and that NAC diminishes bleomycin-induced apoptosis via inhibiting the mitochondrial pathway. We conclude that NAC has negative effects on bleomycin-induced apoptosis in NICCT cells and causes resistance to apoptosis, which is not a desirable effect in the fight against cancer.     

Extinction times and phase transitions for spatially structured closed epidemics

This paper considers the time to extinction for a stochastic epidemic model of SEIR form without replacement of susceptibles. It first shows how previous rigorous results can be heuristically explained in terms of the more transparent dynamics of an approximating deterministic system. The model is then extended to include a host population structured into patches, with weak nearest-neighbour mixing of infection. It is shown, by considering the approximating deterministic system, that the expected time to extinction in a population of n + 1 patches each of size N is of the form a log N + bn, provided that N > N _c where N _c is a critical patch size below which transits are unlikely to occur. This corresponds to the simple decomposition of the time of an epidemic into the time it takes to spread through one patch plus the time it takes to transit to each of n successive patches. Expressions for this threshold and the coefficients of the time to extinction are given in terms of the transmission parameters of infection and the coupling strength between patches. These expressions are compared with numerical results using parameters relevant to a study of phocine distemper virus in North Sea seals, and the agreement is found to be good for large and small N. In the region when N ≈ N _c , where transits may or may not occur, interesting transitional behaviour is seen, leading to a non-monotonicity of the extinction time as a function of N.     

A fine balance: epigenetic control of cellular quiescence by the tumor suppressor PRDM2/RIZ at a bivalent domain in the cyclin a gene

Adult stem cell quiescence is critical to ensure regeneration while minimizing tumorigenesis. Epigenetic regulation contributes to cell cycle control and differentiation, but few regulators of the chromatin state in quiescent cells are known. Here we report that the tumor suppressor PRDM2/RIZ, an H3K9 methyltransferase, is enriched in quiescent muscle stem cells in vivo and controls reversible quiescence in cultured myoblasts. We find that PRDM2 associates with >4400 promoters in G₀ myoblasts, 55% of which are also marked with H3K9me2 and enriched for myogenic, cell cycle and developmental regulators. Knockdown of PRDM2 alters histone methylation at key promoters such as Myogenin and CyclinA2 (CCNA2), and subverts the quiescence program via global de-repression of myogenesis, and hyper-repression of the cell cycle. Further, PRDM2 acts upstream of the repressive PRC2 complex in G₀. We identify a novel G₀-specific bivalent chromatin domain in the CCNA2 locus. PRDM2 protein interacts with the PRC2 protein EZH2 and regulates its association with the bivalent domain in the CCNA2 gene. Our results suggest that induction of PRDM2 in G₀ ensures that two antagonistic programs—myogenesis and the cell cycle—while stalled, are poised for reactivation. Together, these results indicate that epigenetic regulation by PRDM2 preserves key functions of the quiescent state, with implications for stem cell self-renewal.     

TGFβ signaling in male germ cells regulates gonocyte quiescence and fertility in mice

During testis development, proliferation and death of gonocytes are highly regulated to establish a standard population of adult stem spermatogonia that maintain normal spermatogenesis. As Transforming Growth Factor beta (TGFbeta) can regulate proliferation and apoptosis, we investigated its expression and functions during testis development. We show that TGFbeta2 is only expressed in quiescent gonocytes and decreases gonocyte proliferation in vitro. To study the functions of TGFbeta2, we developed conditional mice that invalidate the TGFbeta receptor type II in germ cells. Most of the knock-out animals die during fetal life, but the surviving adults show a reduced pool of spermatogonial stem/progenitor cells and become sterile with time. Using an organ culture system mimicking in vivo development, we show higher proportions of proliferating and apoptotic gonocytes from 13.5 dpc until 1 dpp, suggesting a reduction of germinal quiescence in these animals. Conversely, a 24-hour TGFbeta2-treatment of explanted wild-type testes, isolated every day from 13.5 dpc until 1 dpp, increased the duration of quiescence.These data show that the TGFbeta signaling pathway plays a physiological role during testis development by acting directly as a negative regulator of the fetal and neonatal germ cell proliferation, and indicate that the TGFbeta signaling pathway might regulate the duration of germ cell quiescence and is necessary to maintain adult spermatogenesis.     

Potential role of Noxes in the protection of mucosae: H2O2 as abacterial repellent

Duox proteins are members of the NADPH oxidase (Nox) family and are responsible for hydrogen peroxide (H₂O₂) production by various tissue types including bronchial and intestinal mucosae. The antimicrobial killing role of H₂O₂ in leukocytes and macrophages is generally considered as the paradigm of its function. We investigated here the positive role of H₂O₂ in the prevention of cellular invasion by Salmonella. We show that H₂O₂, under conditions that preserved bacterial growth, has a repellent effect on Salmonella motility on agar plates. In addition, H₂O₂ produced by PCCl3, a rat thyroid cell line, reduces bacterial invasion of the cells by around 40%. To test whether the observed phenotype is attributable to H₂O₂ production, we constructed a CHO stable cell line expressing Duox2 protein at the cell surface (CHO-D2). The transfected cells produce a high amount of H₂O₂. Upon infection with Salmonella, the invasion of CHO-D2 cells was reduced by up to 60%. In both PCCl3 and CHO expressing Duox2 cells, normal invasion was restored upon incubation with catalase. Our data suggest that H₂O₂ at reduced concentrations acts as a repellent for bacteria, keeping them away from cells, a situation that could naturally prevent mucosal cells infection in vivo.     

Enhanced Killing of Acanthamoeba Cysts with a Plant Peroxidase-Hydrogen Peroxide-Halide Antimicrobial System

The activity of H₂O₂ against the resistant cyst stage of the pathogenic free-living amoeba Acanthamoeba was enhanced by the addition of KI and either horseradish peroxidase or soybean peroxidase or, to a lesser degree, lactoperoxidase. This resulted in an increase in the cysticidal activity of 3% (wt/vol) H₂O₂, and there was >3-log killing in 2 h, compared with the 6 h required for comparable results with the peroxide solution alone (P < 0.05). With 2% H₂O₂, enhancement was observed at all time points (P < 0.05), and total killing of the cyst inoculum occurred at 4 h, compared with 6 h for the peroxide alone. The activity of sublethal 1% H₂O₂ was enhanced to give 3-log killing after 8 h of exposure (P < 0.05). No enhancement was obtained when KCl or catalase was used as a substitute in the reaction mixtures. The H₂O₂ was not neutralized in the enhanced system during the experiments. However, in the presence of a platinum disk used to neutralize H₂O₂ in contact lens care systems, the enhanced 2% H₂O₂ system gave 2.8-log killing after 6 h or total cyst killing by 8 h, and total neutralization of the H₂O₂ occurred by 4 h. In contrast, 2% H₂O₂ alone resulted in <0.8-log killing of cysts in the presence of the platinum disk due to rapid (<1 h) neutralization of the peroxide. Our observations could result in significant improvement in the efficacy of H₂O₂ contact lens disinfection systems against Acanthamoeba cysts and prevention of acanthamoeba keratitis.     

ΔNp63α promotes cellular quiescence via induction and activation of Notch3

Genetic analysis of TP63 indicates that ΔNp63 isoforms are required for preservation of self-renewing capacity in the stem cell compartments of diverse epithelial structures; however, the underlying cellular and molecular mechanisms remain incompletely defined. Cellular quiescence is a common feature of adult stem cells that may account for their ability to retain long-term replicative capacity while simultaneously limiting cellular division. Similarly, quiescence within tumor stem cell populations may represent a mechanism by which these populations evade cytotoxic therapy and initiate tumor recurrence. Here, we present evidence that ΔNp63α, the predominant TP63 isoform in the regenerative compartment of diverse epithelial structuresm, promotes cellular quiescence via activation of Notch signaling. In HC11 cells, ectopic ΔNp63α mediates a proliferative arrest in the 2N state coincident with reduced RNA synthesis characteristic of cellular quiescence. Additionally, ΔNp63α and other quiescence-inducing stimuli enhanced expression of Notch3 in HC11s and breast cancer cell lines, and ectopic expression of the Notch3 intracellular domain (N3^ICD) was sufficient to cause accumulation in G₀/G₁ and increased expression of two genes associated with quiescence, Hes1 and Mxi1. Pharmacologic inhibition of Notch signaling or shRNA-mediated suppression of Notch3 were sufficient to bypass quiescence induced by ΔNp63α and other quiescence-inducing stimuli. These studies identify a novel mechanism by which ΔNp63α preserves long-term replicative capacity by promoting cellular quiescence and identify the Notch signaling pathway as a mediator of multiple quiescence-inducing stimuli, including ΔNp63α expression.Key words: p63, Notch, quiescence, stem cell     

Effects of N-acetyl-l-cysteine on bleomycin induced oxidative stress in malignant testicular germ cell tumors

Testicular cancer is a very common cancer in males aged 15–44 years. Bleomycin is used in chemotherapy regimens in the treatment of patients having testicular germ-cell tumor. Bleomycin generates oxygen radicals, induces oxidative cleavage of DNA strand and induces apoptosis in cancer cells. There is no study in the literature investigating effects of N-Acetyl-l-Cysteine (NAC) on bleomycin-induced oxidative stress in testicular germ cell tumors. For this reason, we studied effects of NAC on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testis cancer cells incubated with bleomycin and compared the results with H₂O₂ which directly produces oxidative stress. We determined protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8-isoprostane, lipid hydroperoxide levels and total antioxidant capacity in both testicular cancer cells. Bleomycin and H₂O₂ significantly increased 8-isoprostane, TBARS, protein carbonyl and lipid hydroperoxide levels in NTera-2 and NCCIT cells. Bleomycin and H₂O₂ significantly decreased antioxidant capacity and GSH levels in both cell lines. Co-incubation with NAC significantly decreased lipid hydroperoxide, 8-isoprostane, protein carbonyl content and TBARS levels increased by bleomycin and H₂O₂. NAC enhanced GSH levels and antioxidant capacity in the NTera-2 and NCCIT cells. It can be concluded that NAC diminishes oxidative stress in human testicular cancer cells induced by bleomycin and H₂O₂.     

Mammalian MutY homolog (MYH or MUTYH) protects cells from oxidative DNA damage

MutY DNA glycosylase homologs (MYH or MUTYH) reduce G:C to T:A mutations by removing misincorporated adenines or 2-hydroxyadenines paired with guanine or 8-oxo-7,8-dihydroguanine (8-oxo-G). Mutations in the human MYH (hMYH) gene are associated with the colorectal cancer predisposition syndrome MYH-associated polyposis. To examine the function of MYH in human cells, we regulated MYH gene expression by knockdown or overproduction. MYH knockdown human HeLa cells are more sensitive to the killing effects of H₂O₂ than the control cells. In addition, hMYH knockdown cells have altered cell morphology, display enhanced susceptibility to apoptosis, and have altered DNA signaling activation in response to oxidative stress. The cell cycle progression of hMYH knockdown cells is also different from that of the control cells following oxidative stress. Moreover, hMYH knockdown cells contain higher levels of 8-oxo-G lesions than the control cells following H₂O₂ treatment. Although MYH does not directly remove 8-oxo-G, MYH may generate favorable substrates for other repair enzymes. Overexpression of mouse Myh (mMyh) in human mismatch repair defective HCT15 cells makes the cells more resistant to killing and refractory to apoptosis by oxidative stress than the cells transfected with vector. In conclusion, MYH is a vital DNA repair enzyme that protects cells from oxidative DNA damage and is critical for a proper cellular response to DNA damage.     

N,N-Bis(glycityl)amines as anti-cancer drugs

A series of N,N-bis(glycityl)amines with promising anti-cancer activity were prepared via the reductive amination of pentoses and hexoses, and subsequently screened for their ability to selectively inhibit the growth of cancerous versus non-cancerous cells. For the first time, we show that this class of compounds possesses anti-proliferative activity, and, while the selective killing of brain cancer (LN18) cells versus matched (SVG-P12) cells was modest, several of the amines, including d-arabinitylamine 1a and d-fucitylamine 1g, exhibited low micromolar IC₅₀ values for HL60 cells. Moreover, these two amines showed good selectivity towards HL60 cells when compared to non-cancerous HEK-293 cells. The compounds also showed low micromolar inhibition of the leukaemic cell line, THP-1. The modes of action of amines 1a and 1g were then determined using yeast chemical genetics, whereby it was established that both compounds affect similar but distinct sets of biochemical pathways. Notably purine nucleoside monophosphate biosynthesis was identified as an enriched mechanism. The rapid synthesis of the amines and their unique mode of action thus make them attractive targets for further development as anti-cancer drugs.     

Synthesis and anticancer activity of focused compound libraries from the natural product lead,oroidin

Oroidin (1), (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-4,5-dibromo-1H-pyrrole-2-carboxamide, is a pyrrole alkaloid isolated from the marine sponge Agelas oroides. Routine screening in a panel of twelve cancer cell lines revealed 1 to be poorly cytotoxic with the 50% growth inhibition concentration (GI₅₀) of 42 μM in MCF-7 (breast) cells and 24 μM in A2780 (ovarian) cells and >50 μM in all other cell lines tested. The development of eight focused libraries comprising thirty compounds total identified N-(biphenyl-4-ylmethyl)-1H-pyrrole-2-carboxamide (4l), N-benzyl-4,5-dibromo-1H-pyrrole-2-carboxamide (5a) and N-(biphenyl-4-ylmethyl)-4,5-dibromo-1H-pyrrole-2-carboxamide (5l) as potent inhibitors of cell growth in our panel of cell lines. Of these compounds GI₅₀ values of <5 μM were observed with 4l against HT29 (colon) and SW480 (colon); 5a against HT29; and 5l against HT29, SW480, MCF-7, A431 (skin), Du145 (prostate), BE2-C (neuroblastoma) and MIA (pancreas) cell lines. As a cancer class, colon cancer appears to be more sensitive to the oroidin series of compounds, with analogue 5l being the most active.     

Taking multiple infections of cells and recombination into account leads to small within-host effective-population-size estimates of HIV-1

Whether HIV-1 evolution in infected individuals is dominated by deterministic or stochastic effects remains unclear because current estimates of the effective population size of HIV-1 in vivo, N_e, are widely varying. Models assuming HIV-1 evolution to be neutral estimate N_e∼10²–10⁴, smaller than the inverse mutation rate of HIV-1 (∼10⁵), implying the predominance of stochastic forces. In contrast, a model that includes selection estimates N_e>10⁵, suggesting that deterministic forces would hold sway. The consequent uncertainty in the nature of HIV-1 evolution compromises our ability to describe disease progression and outcomes of therapy. We perform detailed bit-string simulations of viral evolution that consider large genome lengths and incorporate the key evolutionary processes underlying the genomic diversification of HIV-1 in infected individuals, namely, mutation, multiple infections of cells, recombination, selection, and epistatic interactions between multiple loci. Our simulations describe quantitatively the evolution of HIV-1 diversity and divergence in patients. From comparisons of our simulations with patient data, we estimate N_e∼10³–10⁴, implying predominantly stochastic evolution. Interestingly, we find that N_e and the viral generation time are correlated with the disease progression time, presenting a route to a priori prediction of disease progression in patients. Further, we show that the previous estimate of N_e>10⁵ reduces as the frequencies of multiple infections of cells and recombination assumed increase. Our simulations with N_e∼10³–10⁴ may be employed to estimate markers of disease progression and outcomes of therapy that depend on the evolution of viral diversity and divergence.