目的： 尽管过去几十年癌症的化疗取得了很大进步，但晚期非小细胞肺癌的预后仍然较差。表皮生长因子受体酪氨酸激酶抑制剂（epidermal growth factor receptor tyrosine kinase inhibitors，EGFR TKIs）给晚期非小细胞肺癌的患者带来了新的希望。然而，EGFR TKIs的总体效果有限，且不良反应较多，价格也较昂贵。如果能找到EGFR TKIs的疗效预测因子，则该治疗就可以只给予那些最有可能从中获益的人，从而提高成本效果，并使治疗变得更加个体化。 / 已有单组研究在接受EGFR TKIs治疗的患者中对有或没有某个标志物的人的预后进行了比较，发现EGFR基因突变、EGFR基因拷贝数增加、EGFR蛋白表达和KRAS基因突变这4个生物标志物可能能够预测EGFR TKIs的疗效。然而，此类研究的方法学是有缺陷的。要确定以上生物标志物是否有预测作用，应该在评估EGFR TKIs疗效的随机对照试验中作亚组分析，对该治疗在有某个生物标志物及没有某个生物标志物的患者中的疗效进行比较，检测治疗与生物标记物的交互作用。 / 但是，现有的随机对照试验通常样本量较小，统计效能不足，难以从中得到确定的结论。因此，我们做了一个随机对照试验的系统综述，以总结现有的最佳证据，对EGFR TKIs与上述4个生物标志物的交互作用进行评估。 / 方法： 我们检索了PubMed，EMBASE，考科蓝图书馆，中国生物医学文献数据库（中文），万方数据库（中文），美国临床肿瘤学会和欧洲肿瘤学会的会议摘要，以及相关原始研究、系统综述与Meta分析、临床指南、共识及专家意见的参考文献。检索时间截至2012年6月。合格研究为非重复、提供了具体数据且符合下列所有条件的研究：1）研究对象：晚期非小细胞肺癌患者；2）干预措施：EGFR TKIs单药治疗或联合其他药物治疗；3）对照措施：安慰剂对照，空白对照或化疗，或者它们任一种加上干预组的基线治疗；4）结局指标：无进展生存期和/或总生存期；5）研究设计：随机对照试验；6）根据上述任一种或多种生物标志物的状态作了亚组分析。 / 两名研究者平行独立地从合格研究中提取了患者特征、治疗方案、结局、生物标志物分析和方法学质量等方面的资料。对每一个研究，我们都根据生物标志物阳性亚组的风险比（hazard ratio）和阴性亚组的风险比计算了一个风险比之比（ratio of hazard ratios）来测量该标志物对疗效的预测能力或者说治疗与该生物标志物的交互作用。然后，采用随机效应模型对来自不同研究的风险比之比进行Meta分析；采用Cochran Q检验和I²评估研究间的异质性；通过敏感性分析考察原始研究的方法学质量等因素对结果的影响；采用Begg漏斗图和Egger检验来检测发表偏倚存在的可能性。 / 结果： 共有18个合格研究入选。可用于各个生物标志物分析的患者数量从1763到3246不等。原始研究普遍对关于方法学质量的信息报告得不够充分；有的研究可能存在重要偏倚。与安慰剂相比，EGFR TKIs可以有效延长无进展生存期和总生存期，但对总生存期的效果相对较小。除了在EGFR基因突变的患者中EGFR TKIs延长无进展生存期的效果明显好于化疗外，其它情形下，不管是无进展生存期还是总生存期，EGFR TKIs与化疗的效果均相当。 / 以无进展生存期为结局的风险比之比，在EGFR基因突变状态不同的亚组间（野生型亚组为参照）为0.37（95% 置信区间[CI]：0.22-0.60，P < 0.0001），EGFR基因拷贝数状态不同的亚组间（未增加的亚组为参照）为0.72（95% CI：0.52-0.99，P = 0.04），EGFR蛋白表达状态不同的亚组间（无表达的亚组为参照）为0.99（95% CI：0.78-1.26，P = 0.93），KRAS基因突变状态不同的亚组间（野生型亚组为参照）为1.35（95% CI：1.02-1.80，P = 0.04）。这些结果提示EGFR TKIs治疗与EGFR基因突变，EGFR基因拷贝数及KRAS基因突变之间可能存在交互作用。以总生存期为结局的风险比之比，在EGFR基因突变、EGFR基因拷贝数、EGFR蛋白表达及KRAS基因突变状态不同的亚组间分别为0.84（95% CI：0.64-1.11，P = 0.22）、0.92（95% CI：0.69-1.23，P = 0.57）、0.86（95% CI：0.70-1.05，P = 0.14）和1.37（95% CI：0.89-2.10，P = 0.15）。 / 就统计学显著性、异质性和稳定性而言，关于其它3个生物标志物的结果不如EGFR基因突变的相关结果确定，关于总生存期的结果不如无进展生存期的相关结果确定。没有证据表明本研究中存在发表偏倚。 / 结论： EGFR基因突变可用于确定哪些患者更有可能从EGFR TKIs治疗中获益。EGFR基因拷贝数增加和KRAS基因突变可能也有类似用途，但它们与治疗的交互作用是独立存在的还是由于它们与EGFR基因突变的相关性而获得的，目前尚不清楚。在EGFR野生型的患者中，选择化疗似乎比EGFR TKIs更好，因为它的副作用相对较少，且更为便宜。 / 本研究的结果为当前的临床指南提供了全面的证据支持。其它3个标志物在EGFR野生型患者中的预测价值可能还值得进一步的探讨，但我们更建议未来的研究在探讨治疗与生物标志物的交互作用时进行多因素分析。 / Objective: Despite the many new progresses in chemotherapy, the prognosis of advanced non-small cell lung cancer (NSCLC) remains poor. The introduction of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) seems to offer new promises for advanced NSCLC patients. However, EGFR TKIs have a limited overall efficacy, clear adverse events and large costs. It has become particularly appealing to identify, through new biomarkers, patients who are more likely to benefit from the treatment so that the treatment can be more personalized and effective. / EGFR mutations, EGFR gene copy number gain, EGFR protein expression and KRAS mutations were indicated as potential predictive biomarkers for the efficacy of the treatment in single-arm studies that compared survival of treated patients with and without a biomarker. However, such comparisons are flawed and the appropriate study design to evaluate the value of a biomarker in predicting efficacy which is known as interaction in epidemiology is the randomized controlled trial with stratified analysis that compared the efficacy of EGFR TKIs between patients with and without the biomarker. / As trials in this field are usually small in sample size and insufficiently powered for drawing a robust conclusion, we conducted this systematic review to summarize the evidence from all relevant randomized controlled trials that have data for investigating the interaction between EGFR TKIs and the 4 biomarkers. / Methods: PubMed, EMBASE, the Cochrane Library, Chinese Biomedical Literature Database (in Chinese), Wanfang Data (in Chinese), the abstracts of conferences of the American Society of Clinical Oncology and European Society of Medical Oncology, the reference list of relevant original studies, systematic reviews and meta-analyses, guidelines, consensus, and expert opinions were searched up to June 2012. / Eligible studies had to be non-duplicate, extractable studies meeting all the following criteria: 1) Population: patients with advanced NSCLC; 2) Intervention: EGFR TKIs alone or EGFR TKIs plus other treatments; 3) Control: placebo, no treatment, or chemotherapy, with or without the baseline treatments in the intervention arm; 4) Outcome: progression-free survival and/or overall survival; 5) Study design: randomized controlled trial; 6) Subgroup analyses were conducted according to the status of one or more of the 4 biomarkers. / Data on patients’ characteristics, treatment protocols, outcomes, biomarker analysis and methodological quality were extracted by two researchers independently. Within a study, we defined the measure of the value of a biomarker in predicting efficacy or biomarker-treatment interaction as the hazard ratio in patients with the biomarker relative to that in those without the marker. The ratio of hazard ratios from relevant studies was then combined by using the random-effect model. / Heterogeneity among studies was assessed by the Cochran’ Q test and I². Sensitivity analyses were conducted to examine the impact of factors such as methodological quality on the results. Begg’s funnel plots and Egger’s tests were used to examine the possibility of publication bias. / Results: Eighteen studies were included. The number of patients available for analyses on different biomarkers varied from 1,763 to 3,246. Data on the methodological quality of included studies are generally under-reported. Some studies seemed to have important biases. EGFR TKIs are in general effective in increasing progression-free and overall survival as compared with placebo although the effect size is smaller for overall survival than for progression free survival. EGFR TKIs are comparable to chemotherapy in their effect in prolonging both progression-free and overall survival, except in EGFR mutation group in which EGFR TKIs seem much more effective than chemotherapy in prolonging progression-free survival. / Importantly, for progression-free survival, the summary ratio of hazard ratios was 0.37 (95% confidence interval [CI]: 0.22-0.60, P < 0.0001) for EGFR mutations (versus wild-type), 0.72 (95% CI: 0.52-0.99, P = 0.04) for EGFR gene copy number gain (versus no gain), 0.99 (95% CI: 0.78-1.26, P = 0.93) for EGFR protein expression (versus negative), and 1.35 (95% CI: 1.02-1.80, P = 0.04) for KRAS mutations (versus wild-type), indicating interaction may exist between EGFR TKIs and EGFR mutation, EGFR gene copy number and KRAS mutations. For overall survival, the summary ratio of hazard ratios for EGFR mutations, EGFR gene copy number gain, EGFR protein expression and KRAS mutations was 0.84 (95% CI: 0.64-1.11, P = 0.22), 0.92 (95% CI: 0.69-1.23, P = 0.57), 0.86 (95% CI: 0.70-1.05, P = 0.14) and 1.37 (95% CI: 0.89-2.10, P =0.15), respectively. / In general, the results on EGFR gene copy number gain, KRAS mutations and EGFR protein expression were less certain than those on EGFR mutations in terms of statistical significance, consistency and robustness, and the results on overall survival were less certain than those on progression-free survival. Publication bias did not seem present in the study. / Conclusions: EGFR mutations and possibly EGFR-GCN and KRAS mutations can help identify who are more likely to benefit from EGFR TKIs treatment. However, it is not clear whether the interaction with EGFR-GCN and KRAS mutations are independent or obtained through their relation with EGFR mutations. Furthermore, in EGFR wild-type patients, given that chemotherapy is cheaper and of fewer side effects, chemotherapy seems clearly a better choice than EGFR TKIs. / Our findings provided the most comprehensive evidence for the recommendations of current guidelines. Although the predictive value of the other 3 biomarkers in wild-type EGFR patients may be worth further investigation, we suggest that multivariate analyses are explored in future studies of biomarker-treatment interactions. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yang, Zuyao. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 88-104). / Abstracts also in Chinese. / Yang, Zuyao.

Chemotherapy in childhood cancer patients is associated with reduced bone density that can result in osteoporotic fracture in survivors. A significant proportion of paediatric patients experience a reduction in plasma 25 - hydroxyvitamin D3 [ 25 ( OH ) D3 ] and 1,25 - dihydroxyvitamin D3 [ 1,25 ( OH ) 2D3 ] levels during treatment, the basis of which is unknown. A balance between the bioactivation and degradation of 1,25 ( OH ) 2D3 is responsible for maintaining homoeostatic levels of 1,25 ( OH ) 2D3 at the correct set - point. Whereas the cytochrome P450 enzyme, CYP27B1 ( 25 - hydroxyvitamin D3 1 α - hydroxylase ), catalyses the hydroxylation of the precursor 25 ( OH ) D3 to generate 1,25 ( OH ) 2D3, catabolic inactivation and cleavage of 1,25 ( OH ) 2D3 is achieved by the mitochondrial cytochrome P450 enzyme, 25 - hydroxyvitamin D3 24 - hydroxylase ( CYP24 ), which is highly expressed in bone and kidney cells. Since many of the signalling pathways which regulate the expression of CYP24 are also activated by chemotherapeutic drugs, we hypothesised that the drugs could cause the degradation of plasma 25 ( OH ) D3 and 1,25 ( OH ) 2D3 by increasing CYP24 expression, the principal means of facilitating the bio - inactivation and degradation of plasma 25 ( OH ) D3 and 1,25 ( OH ) 2D3. Using the kidney cell - lines, COS - 1 and HEK293T cells, we now report that chemotherapeutic drugs, represented by daunorubicin hydrochloride ( an anthracycline antibiotics ), etoposide and vincristine sulphate ( vinca alkaloids and related compounds ) and cisplatin ( an alkylating agent ), were able to enhance CYP24 promoter activity in kidney cell lines transfected with a CYP24 promoter - luciferase construct, either by themselves or in the presencedaunorubicin hydrochloride and etoposide, two of the strongest inducers of CYP24 promoter activation under our experimental conditions, demonstrate that these drugs acted in a concentration - dependent manner. In addition to stimulating promoter activity on their own, the drugs also amplified the induction of the CYP24 promoter by 1,25 ( OH ) 2D3. Synergistic increases were generally observed when the cells were treated simultaneously with 1,25 ( OH ) 2D3 and a drug. The two kidney cell lines generally responded in a similar manner when challenged with the drugs, either in the presence or absence of 1,25 ( OH ) 2D3. Interestingly, the hydroxylated derivative of daunorubicin hydrochloride, doxorubicin hydrochloride which is also a commonly used chemotherapeutic drug, had no effect of promoter activity. Further studies with daunorubicin hydrochloride demonstrated that the effects of the drug per se were not mediated by oxidative stress and the vitamin D receptor was not required for daunorubicin hydrochloride per se to stimulate CYP24 promoter activity. However, daunorubicin hydrochloride caused a modest increase in the expression of the vitamin D receptor and this could contribute to its synergistic activity with 1,25 ( OH ) 2D3. In the presence of etoposide, there was also a tendency for the kidney cells to express higher levels of the vitamin D receptor. A key role for the extracellular signal - regulated protein kinase ( ERK ) 1, ERK2 and ERK5 mitogen - activated protein ( MAP ) kinases was demonstrated for the inductive action of daunorubicin hydrochloride and etoposide, with CYP24 promoter - specific transcription factors located in the first - 298bp being likely targets of the ERK activity. Studies with a dominant negative mutant of MKK4, one of the two immediate upstream activators of the c - jun N - terminal kinase isoforms, demonstrated that this MAP kinase also played a crucial role in inductive actions of the of 1,25 ( OH ) 2D3. Dose - response studies with drugs. Consistent with their use in anti - cancer therapy, all of the above drugs killed the human promyelocytic HL60 leukaemic cells at very low concentrations but had no effect on the viability of kidney or liver cells, either at concentrations used in our experiments or at higher levels. Our data provide novel biochemical evidence that some of the commonly used chemotherapeutic drugs could cause an increase in the transcriptional activation of the promoter, most likely via the MAP kinases activating the transcription factors which bind to the CYP24 promoter. Such an effect could contribute to the reduction in plasma 25 ( OH ) D3 and 1,25 ( OH ) 2D3 in some of the patients undergoing chemotherapy. / Thesis (Ph.D.)--School of Paediatrics and Reproductive Health, 2005.

Cancer has been a disease, which has generated intense research interest for many years. Misexpression of two oncoproteins, TAL 1 and LMO 1, has been found to help induce a particular type of leukemia, called T-cell acute lymphoblastic leukemia (T-ALL). Presently, it is not completely understood how these proteins induce leukemogenesis or what other cellular proteins they interact with to drive this progression. In this study, a series of experiments were conducted to identify downstream targets of TALI and LMO1. Using retroviral gene transfer, both genes were introduced, either singly or in combination, into a murine T-cell line called AKR-DP-603. Empty vectors were introduced as controls. In order to assay the effects of TALI and LMO I expression on expression of other proteins, a series of Western blots were completed on all populations of engineered cells. It was determined that there were differences in expression of Bcl-2 and p16 as indicated by differences in band intensities on the blots. This is important because it implies an effect on protein levels by TAL 1 and LMO 1. However, there were no differences in protein expression levels for Bax or cyclin D1. This suggests that TAL1 and LMOI do not have any regulatory effects on these proteins. In addition, apoptotic assays were completed on all populations of cells. The results of both a TUNEL assay and ethidium bromide/acridine orange staining protocol showed TAL1- and LMO1expressing cells to have an increase in cell survival under starvation conditions and a lower frequency of apoptosis. Statistical analysis verified significant difference in the apoptosis assays. The data suggests an up-regulation of anti-apoptotic proteins. The finding of this research allow a clearer understanding of the process of leukemogenesis and may lead to a development of better cancer treatments. / Department of Biology

Hu, Baoguang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 121-143). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

The crystal structures of two thermostable carboxylesterase Est30 and Est55 from Geobacillus stearothermophilus were determined to help understand their functions and applications in industry or medicine. The crystal structure of Est30 was determined at 1.63 Å resolution by the multiple anomalous dispersion method. The two-domain Est30 structure showed a large domain with a modified alpha/beta hydrolase core including a seven, rather than an eight-stranded beta sheet, and a smaller cap domain comprising three alpha helices. A 100 Da tetrahedral ligand, propyl acetate, was observed to be covalently bound to the side chain of Ser94 in the catalytic triad. This ligand complex represents the first tetrahedral intermediate in the reaction mechanism. Therefore, this Est30 crystal structure will help understand the mode of action of all enzymes in the serine hydrolase superfamily. Est55 is a bacterial homologue of the mammalian carboxylesterases involved in hydrolysis and detoxification of numerous peptides and drugs and in prodrug activation. Est55 crystals were grown at pH 6.2 and pH 6.8 and the structures were determined at resolutions of 2.0 and 1.58 Å respectively. Est55 folds into three domains, a catalytic domain, an α/β domain and a regulatory domain. This structure is in an inactive form; the side chain of His409, one of the catalytic triad residues, is pointing away from the active site. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the entry of substrate to its binding site. This structure suggested a self-inactivation mechanism, however, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side chains at this position were favorable, while polar serine was unfavorable for enzyme activity. Both Est30 and Est55 were shown to hydrolyze the prodrug CPT-11 into the active form SN-38. Therefore, Est30 and Est55 are potential candidates for use with irinotecan in cancer therapy. The catalytic efficiency (kcat/Km) of Est30 is about 10-fold lower than that of Est55. The effects of the Cys408 substitutions on Est55 activity differed for the two substrates, p-NP butyrate and CPT-11. Mutant C408V may provide a more stable form of Est55.

In the 1980's, Nicholas Farrell developed a range of structurally distinct multinuclear Pt complexes that form long-range interstrand crosslinks (IXLs) in DNA. The dinuclear complex [{trans-PtCl2(NH3)}2-µ-(H2N(CH2)6NH2)]2+ (1,1/t,t) was the first of this series to show promising results, however, it was the trinuclear complex [{trans-PtCl2(NH3)}2-µ-trans-Pt(NH3)2(H2N(CH2)6NH2)2]4+ (1,0,1/t,t,t or BBR3464) that was chosen for clinical trials based on significantly increased cytotoxicity compared to 1,1/t,t and cisplatin. Molecular biology experiments have shown that 1,1/t,t exclusively forms IXLs in DNA in the 5'¿ 5' direction, whilst 1,0,1/t,t,t can form IXLs in both the 5'¿5' and 3'¿3' directions. Previously, 2D [1H,15N] HSQC NMR has been used to study the formation of 5'–5' 1,4–GG IXLs. The formation of 3'–3' 1,4–GG IXLs have been studied as part of this thesis. More recently, Pt complexes such as [{trans–PtCl2(NH3)}2{H2N(CH2)6(NH2(CH2)2NH2)(CH2)6NH2}]4+ (1,1/t,t–6,2,6) and [{trans–PtCl2(NH3)}2{H2N(CH2)6(NH2)(CH2)6NH2}]3+ (1,1/t,t–6,6), where the charged central Pt moiety of 1,0,1/t,t,t is replaced by a polyamine linker, have been developed in the Farrell group and show increased potency compared to 1,0,1/t,t,t. The complex 1,1/t,t 6,2,6 is a lead candidate currently undergoing Phase I clinical trials. Prior to the work presented in this thesis, little was known about the aquation chemistry or kinetics of DNA binding of these novel complexes. Reported in Chapter 3 is the study of the formation of 3'–3' 1,4–GG IXLs by both 1,0,1/t,t,t and 1,1/t,t in the duplex 5' {d(TATACATGTATA)2} (33–14XL) (pH 5.4, 298K). A combination of 1D 1H and 2D [1H, 15N] HSQC NMR experiments was used to directly compare the results with the stepwise formation of the 5'–5' 1,4–GG IXL with the previously studied duplex, 5' {d(ATATGTACATAT)2} (55–14XL), under the same conditions. Preassociation as well as aquation were similar, however, differences were observed at the monofunctional binding step with evidence for numerous monofunctional adducts. Both reactions did not yield a single 3'–3' 1,4–GG IXL, rather several adducts that could not be characterised. Molecular dynamics simulations of the 3'–3' 1,4–GG IXLs showed highly distorted lesions that may have implication in cellular repair processes.

BRCA1 and BRCA2 are gene mutations that drastically increase chances of developing breast and ovarian cancers, up to 20-fold, for women. A genetic blood test is used to detect BRCA mutations. Though these mutations occur in one of every 400 in the general population (excluding Ashkenazi Jewish ethnicity), they are present in most cases of hereditary breast and ovarian cancer patients. Hence, it is common practice for the physicians to require genetic testing for those that fit the rules as recommended by the National Cancer Comprehensive Network. However, data from the Myriad Laboratory, the only provider of the test until 2013, show that over 70 percent of those tested are negative for BRCA mutations [1]. As there are significant costs and psychological trauma associated with having to go through the test, there is a need for more comprehensive rules for determining who should be tested. Once the presence of BRCA is identified via testing, the next challenge for both mutation carriers and their physicians is to select the most appropriate types and timing of intervention actions. Organizations such as the American Cancer Society suggest drastic intervention actions such as prophylactic surgeries and intense breast screenings. These actions vary significantly in their cost, cancer incidence prevention ability, and can have major side effects potentially resulting in reproduction inability or death. Effectiveness of these intervention actions is also age dependent. In this dissertation, both an analytical and an optimization framework are presented. The analytical framework uses supervised machine learning models on extended family history of cancers, and personal and medical information from a recent nationwide survey study of women who have been referred for genetic testing for the presence of a BRCA mutation. This framework provides the potential mutation carriers as well as their physician with an estimate of the likelihood of having the mutations. The optimization framework uses a Markov decision process (MDP) model to find cost-optimal and/or quality-adjusted life years (QALYs) optimal intervention strategies for those tested positive for a BRCA mutation. This framework uses a dynamic approach to address this problem. The decisions are made more robust by considering the variation in estimates of the transition probabilities by using a robust version of the MDP model. This research study delivers an innovative decision support tool that enables physicians and genetic consultants predict the population at high risk of breast and ovarian cancers more accurately. For those identified with presence of the BRCA mutation, the decision support tool offers effective intervention strategies considering either minimizing cost or maximizing QALYs to prevent incidence of cancers.

Le traitement du cancer par thérapie génique nécessite d’une part des gènes suicides efficaces et, d’autre part, l’adressage spécifique de ces gènes aux cellules cancéreuses. J'ai d'abord caractérisé un nouveau gène suicide dérivé de la désoxycytidine kinase humaine (dCK) : le M36. Comparé à la dCK, le M36 permet une meilleure sensibilisation des certaines cellules cancéreuses aux traitements avec différents chimiothérapeutiques comme la gemcitabine et la cytarabine. Ces résultats sont particulièrement encourageants pour l'élimination des cellules cancéreuses résistantes à ces traitements du fait d’un défaut de la dCK. Dans une deuxième partie, je me suis intéressée à l'adressage spécifique des transgènes aux cellules cancéreuses par les vecteurs lentiviraux. J'ai travaillé à la preuve de concept qu’une enveloppe (Env) VIH modifiée peut permettre un tel ciblage. J'ai généré une Env qui a fortement diminué son tropisme naturel et qui comporte un motif liant le marqueur tumoral modèle HER2. / Cancer gene therapy requires the use of an effective suicide gene and the specific targeting of cancer cells. In my PhD work, I have first characterized a new potential suicide gene derived from human deoxycytidine kinase (dCK): M36. Compared to dCK, M36 improves sensitization of certain cancer cells to treatment with chemotherapeutic compounds as gemcitabine and AraC. These results are particularly encouraging for the elimination of cancer cells resistant to the treatment because of a defect with dCK. In a second part, I have worked at the proof of concept that a modified HIV envelope can allow specific targeting of cancer cells by lentiviral vectors. During this work, I have generated a CD4i envelope with a strongly diminished natural tropism and that carries a motif known to bind the model cell surface cancer marker HER2. This envelope constitutes a good starting material to be improved by evolution in cell culture to obtain specific targeting of HER2+ cells.

Indiana University-Purdue University Indianapolis (IUPUI) / MicroRNAs (miRNA) are short, 18-24 nucleotide long noncoding RNAs. These small RNAs, which are initially transcribed in the nucleus, are transported into the cell cytoplasm where they regulate protein translation either through direct cleavage of mRNA, or indirect inhibition through binding to mRNA and disrupting the protein translation machinery. Recently, miRNAs have gained much attention due to their implication in numerous diseases and cancers. It has been found that heightened or lowered levels of miRNA in diseased cells vs. healthy cells are linked to disease progression. It is therefore immensely important to be able to detect these small molecules. Current detection methods of Northern blotting, microarrays, and qRT-PCR suffer from drawbacks including low sensitivity, a lack of simplicity, being semi-quantitative in nature, time-consuming, and requiring expensive instruments. This work aims to develop novel miRNA technologies which will address these above problems. Bioluminescent labels are promising alternatives to current methods of miRNA detection. Bioluminescent labels are relatively small, similar in size to fluorescent proteins, and they emit very intense signals upon binding to their substrate. Bioluminescent labels are advantageous to fluorescent labels in that they do not require an external excitation source, rather, the excitation energy is supplied through a biochemical reaction. Therefore, background signal due to excitation is eliminated. They also have the advantage of being produced in large amounts through bacterial expression. Four miRNA detection methods are presented which utilize luminescence-based methods. Three employ Renilla luciferase, a bioluminescent protein, and one is based on fluorescence. The presented methods are capable of detecting miRNA from the picomole (nanomolar) level down to the femtomole (picomolar) level. These methods are rapid, sensitive, simple, and quantitative, can be employed in complex matrices, and do not require expensive instruments. All methods are hybridization-based and do not require amplification steps.

Wild-type viruses with intrinsic oncolytic capacity in human includes DNA viruses like some autonomous parvoviruses and many RNA viruses. Recent advances in molecular biology have allowed the design of several genetically modified viruses, such as adenovirus and herpes simplex virus that specifically replicate in, and kill tumor cells. However, still several hurdles regarding clinical limitations and safety issues should be overcome before this mode of therapy can become of clinical relevance. It includes limited virus spread in tumor masses, stability of virus in the blood, trapping within the liver sinusoids, transendothelial transfer, and/or vector diffusion of viral particles to tumor cells, limited tumor transduction, immune-mediated inactivation or destruction of the virus. For replication-competent vectors without approved antiviral agents, suicide genes might be used as fail-safe mechanism. Cancer stem cells are a minor population of tumor cells that possess the stem cell property of self-renewal. Therefore, viruses that target the defective self-renewal pathways in cancer cells might lead to improved outcomes.<p>In this thesis, data we generated in the field of oncolytic autonomous parvoviruses are presented.<p>We replaced capsid genes by reporter genes and assessed expression in different types of human cancer cells and their normal counterparts, either at the level of whole cell population, (CAT ELISA) or at the single cell level, (FACS analysis of Green Fluorescent Protein). Cat expression was substantial (up to 10000 times background) in all infected tumor cells, despite variations according to the cell types. In contrast, no gene expression was detected in similarly infected normal cells, (with the exception of an expression slightly above background in fibroblasts.). FACS analysis of GFP expression revealed that most tumor cells expressed high level of GFP while no GFP positive normal cells could be detected with the exception of very few (less than 0.1%) human fibroblast cells expressing high level of GFP. We also replace capsid genes by genes coding for the costimulatory molecules B7-1 and B7-2 and show that, upon infection with B7 recombinant virions, only tumor cells display the costimulatory molecules and their immunogenicity was increased without any effect on normal cells. Using a recombinant MVM containig the Herpes Simplex thymidine kinase gene, we could get efficient killing of most tumor cell types in the presence of ganciclovir, whithout affecting normal proliferating cells. We also produced tetracycline inducible packaging cell lines in order to improve recombinant vectors yields. The prospects and limitations of these different strategies will be discussed.<p>An overview is given of the general mechanisms and genetic modifications by which oncolytic viruses achieve tumor cell-specific replication and antitumor efficacy. However, as their therapeutic efficacy in clinical trials is still not optimal, strategies are evaluated that could further enhance the oncolytic potential of conditionally replicating viruses in conjunction with other standard therapies. <p>Another exciting new area of research has been the harnessing of naturally tumor-homing cells as carrier cells to deliver oncolytic viruses to tumors. The trafficking of these tumor-homing cells (stem cells, immune cells and cancer cells), which support proliferation of the viruses, is mediated by specific chemokines and cell adhesion molecules and we are just beginning to understand the roles of these molecules. Finally, we will explore some ways deserving further study in order to be able to utilize various oncolytic viruses for effective cancer treatment. <p><p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished