Strategies for gene cloning

Cancer, including genitourinary malignancy, is a consequence of accumulated genetic aberrations in genes involved in crucial regulatory pathways. The result is a deregulation of cellular behaviour, leading to neoplastic transformation, uncontrolled cell proliferation and acquisition of metastatic ability. The development and perfection of techniques in the field of recombinant DNA technology, gene cloning, (differential) analysis of gene expression, and sequencing of genes and proteins have provided a wealth of information about the genetic aberrations associated with cancer development. This “recombinant DNA and gene cloning” technology and the recently developed DNA chip technology may provide new molecular diagnostic tools. Furthermore, the technology of gene cloning in combination with the progress in in vivo gene delivery techniques offers new treatment modalities, like gene therapy, additional to conventional therapies. This review is intended to provide a general introduction to the fundamentals or strategies of recombinant DNA and gene cloning techniques as a basis for understanding the rapidly expanding range of new diagnostic and therapeutic opportunities. Some illustrative examples are provided, addressing basic and biomedical research and possible clinical applications in genitourinary oncology. Received: 16 March 1998 / Accepted 29 July 1998     

RNA interference: natural, experimental, and clinical roles in cancer biology

The old idea of using antisense RNA to block messenger RNA has recently led to powerful new techniques for knocking down expression of individual protein-coding genes. The simplicity and general applicability of these new methods for RNA interference (RNAi) have turned them into fundamental tools in molecular and cellular biology, with more than 5000 publications using them during the few years since they were developed. These experimental methods are now known to exploit fundamental cellular processes that regulate differentiation via genomically encoded RNAi sequences known as microRNAs (miRNAs); changes in endogenous microRNA regulation have now been implicated in oncogenesis. Clinical trials based on local delivery of interfering RNA have already begun. More general methods for safe and effective delivery of interfering RNA to intact organisms are being developed, which could open the way to widespread clinical applications. Because RNAi can provide selective knockdown of almost any protein, it may soon provide an approach to individualized cancer therapy.     

The art of surgery in the 21st century: based on natural sciences and new ethical dimensions

Background and aims In the future, new surgical techniques will only be introduced in clinical practice if evidence-based results—frequently the results of controlled clinical trials—are presented. Unlike any other medical discipline, surgeons provide their diagnostic and operative skills through the surgeons’ hand and the use of technical equipment, which ranges from instruments and devices employed during operation to the use of surgical robots. Results Analysing the fundaments of surgery on the turn of the century, there is only a little doubt about the increasing impact of data deriving from natural sciences on knowledge in medicine and management of diseases. The natural scientific method of detecting, measuring, and verifying facts is the methodological basis of surgery as well. The autonomy of the surgeon’s clinical decision making is significantly restricted by the definition of guidelines. They shift the decision from a single patient to a collective panel. Patient safety and the efficiency of new treatment modalities compared with previous standards are the criteria for the judgement of innovative surgery today. The communication and interaction between surgeon-scientist and patients is guaranteed legally by written consensus. But beside of the high probability of benefit from therapy and written consensus, the surgeon–patient relation is determined by these factors: limitation of time for care of an individual patient, increase of time for administration and documentation, increase of bureaucratic barriers for medical research, and health cost constraints. Conclusion The medical mandate to cure a sick patient is an individual mandate to take action. Measures, numbers, and images are only preconditions for a surgeon’s action in daily clinical work; they can never replace it. The call for an ethical imperative in scientific surgery that is dependent on technology is justified when the state of science and uncritical use of surgical skills and financial constraints have major impact on providing medical care.     

Survival Among Patients with Platinum Resistant, Locally Advanced Non-Small Cell Lung Cancer Treated with Platinum-Based Systemic Therapy

Background  

Recent adjuvant chemotherapy trials after resection of stage II and III non-small cell lung cancer (NSCLC) have identified important survival differences among patients with immunohistochemical evidence suggesting platinum resistance. No clinical information exists regarding the impact upon survival of patients treated with platinum agents who exhibit cellular evidence of their tumors’ resistance to platinum. We evaluated the utility of the extreme drug resistance (EDR) assay to predict mortality among a consecutive group of stage II through IV NSCLC patients receiving adjuvant or definitive platinum-based chemotherapy after resection or surgical biopsy.     

Defining the cancer master switch

Background  

Recent research has focused on signaling cascades and their interactions yielding considerable insight into which genetic pathways are targeted and how they tend to be altered in tumors. Therapeutic interventions now can be designed based on the knowledge of pathways vital to tumor growth and survival. These critical targets for intervention, master switches for cancer, are termed so because the tumor attempts to “flip the switch” in a way that promotes its survival, whereas molecular therapy aims to “switch off” signals important for tumor-related processes.     

Biomarkers for breast cancer

Molecular biomarkers for breast cancer are of several types. Risk biomarkers are those associated with increased cancer risk and include mammographic abnormalities, proliferative breast disease with or without atypia, family clustering and inherited germ-line abnormalities. Surrogate endpoint biomarkers are tissue, cellular or molecular alterations that occur between cancer initiation and progression. These biomarkers are utilized as endpoints in short-term chemoprevention trials. Prognostic biomarkers provide information regarding outcome irrespective of therapy, while predictive biomarkers provide information regarding response to therapy. Candidate prognostic biomarkers for breast cancer include elevated proliferation indices such as Ki-67 and proliferating cell nuclear antigen (PCNA); ER and PR overexpression; markers of oncogene overexpression such as c-erbB-2, TGF-a and EGFr; indicators of apoptotic imbalance including overexpression of bcl-2 and an increased bax/bcl-2 ratio; markers of disordered cell signaling such as p53 nuclear protein accumulation; alteration of differentiation signals such as overexpression of c-myc and related proteins; loss of differentiation markers such as TGF-b II receptor and retinoic acid receptor; and alteration of angiogenesis proteins such as VEGF overexpression. As our knowledge regarding molecular biomarkers for breast cancer increases, prognostic indices will be developed that combine the predictive power of individual molecular biomarkers with specific clinical and pathologic factors.     

Auswahlverfahren wissenschaftlicher Beiträge zum DGU-Kongress 2012

Advanced prostate cancer remains dependent on androgens and signaling through the androgen receptor despite castrate levels of testosterone defined as testosterone levels <1.7 nmol/l. Ketoconazole, a nonspecific inhibitor of androgen synthesis, has been tested in clinical trials and showed clinical activity; however, high doses are needed which are associated with significant sides effects, mainly neurotoxicity, gastrointestinal intolerance, and liver toxicity. Abiraterone acetate is an irreversible inhibitor of two key enzymes of androgen synthesis, 17a-hydroxylase and 17,20-lyase, and has been tested in a randomized phase III study in patients with castration-resistant prostate cancer who progressed after chemotherapy. Abiraterone plus prednisone resulted in a significant overall survival benefit of 4.6 months compared to prednisone alone. Abiraterone was well tolerated, with mostly mild or moderate side effects consistent with secondary mineralocorticoid excess, namely fluid retention, hypokalemia, and hypertension. Abiraterone plus prednisone is considered a new standard therapy option for patients with castration-resistant prostate cancer who progressed after chemotherapy.     

TGF-β mediated DNA methylation in prostate cancer

Almost all tumors harbor a defective negative feedback loop of signaling by transforming growth factor-β (TGF-β). Epigenetic mechanisms of gene regulation, including DNA methylation, are fundamental to normal cellular function and also play a major role in carcinogenesis. Recent evidence demonstrated that TGF-β signaling mediates cancer development and progression. Many key events in TGF-β signaling in cancer included auto-induction of TGF-β1 and increased expression of DNA methyltransferases (DNMTs), suggesting that DNA methylation plays a significant role in cancer development and progression. In this review, we performed an extensive survey of the literature linking TGF-β signaling to DNA methylation in prostate cancer. It appeared that almost all DNA methylated genes detected in prostate cancer are directly or indirectly related to TGF-β signaling. This knowledge has provided a basis for our future directions of prostate cancer research and strategies for prevention and therapy for prostate cancer.     

Prostate cancer genomics

The molecular processes contributing to cancer of the human prostate gland are under intensive investigation. Methods used for discovering genetic alterations involved in prostate neoplasia include family studies designed to map hereditary disease loci, chromosomal studies to identify aberrations that may locate oncogenes or tumor suppressor genes, and comprehensive gene expression studies. These studies determine how various molecular signaling pathways influence or reflect the process of carcinogenesis. However, a comprehensive overview of the cell is necessary to understand all of the dynamic interactions between genes, their protein products, and the network of cellular processes resulting in tumorigenesis. Unraveling the complexity of these systems in a timely manner involves the integration of computers, miniaturization, and automation into molecular biology. New biotechnologies such as the development of automated DNA sequencing and complementary DNA microarrays allow for a systematic, “discoverydriven” approach. These and other technologies afford a comprehensive view of biology and pathology that have the potential to fully characterize the processes involved in neoplasia and therefore provide potential targets for the therapy of prostate and other cancers.     

Development and Trends of Surgical Modalities for Breast Cancer in China: A Review of 16-Year Data

Background Surgery is the most important treatment for nonmetastatic breast cancer; however, the utilization of modern surgical techniques in management of breast cancer in mainland China has not been reported. Methods The medical records of 5887 consecutive breast cancer patients treated surgically in the past 16 years were reviewed retrospectively; the utilization of different surgical modalities and associated clinical outcomes were analyzed. Results Median age of all patients was 50 (range 16–92). About 1015 patients were staged as 0–I, 3569 stage II, 517 stage III, and 786 cases could not be staged. Extensive radical mastectomy (ERM), radical mastectomy (RM), modified radical mastectomy (MRM), simple mastectomy (SM), and breast-conserving surgery (BCS) were used in 8%, 27.2%, 55.7%, 1.5%, and 6.3% of patients, respectively. In addition, 1.3% of patients received breast reconstruction. The proportion of early-stage breast cancer increased, and the surgery patterns varied. MRM gradually replaced ERM and RM. The prevalence of BCS began to increase from the mid-1990s and currently represents about 12%. The prevalence of reconstruction also increased and now accounts for 5%. Age, pathologic pattern, and TNM staging affected the choice of surgery modalities markedly. Although patients receiving RM/ERM had worse survival than those receiving BCS/MRM, the survival outcomes of these four groups were similar in the early-stage population. Conclusions MRM remains the most-used surgical modality in operable breast cancer, although the utilization of BCS for early-stage disease has increased rapidly in last decade. Reconstruction following mastectomy as an alternative to BCS is available. Breast-conserving therapy (BCT) and MRM provide similar local controls and long-term survival for breast cancer. Selection of appropriate candidates for a certain surgery requires an assessment of the patient’s age and clinical and pathological characteristics of the tumor.     

Novel quinazoline HMJ‐30 induces U‐2 OS human osteogenic sarcoma cell apoptosis through induction of oxidative stress and up‐regulation of ATM/p53 signaling pathway

Human osteogenic sarcoma is the most common primary bone tumor. Despite of the success of frontline therapy, about 40% of patients have disease progression and further therapy is palliative and toxic. In this study, we developed a novel quinazoline HMJ‐30 to investigate the cell growth inhibition and apoptotic responses in U‐2 OS human osteogenic sarcoma cells. Our results demonstrated that HMJ‐30 significantly reduced cell viabilities of U‐2 OS, HOS, and 143B cells in a dose‐dependent manner, but it exhibited low cytotoxicity in normal hFOB cells. HMJ‐30 induced DNA damage and apoptosis in U‐2 OS cells as revealed by morphologic changes, comet assay and DAPI staining. Immuno‐staining, colorimetric assays, and Western blotting analyses indicated that activities of caspase‐8, caspase‐9, and caspase‐3 and the levels of Bcl‐2 family‐related proteins (Bcl‐2, Mcl‐1, Bax, BAD, and t‐Bid) were altered in HMJ‐30‐treated U‐2 OS cells. Pretreatment of cells with caspase‐8, ‐9, and ‐3 specific inhibitors significantly reduced the cell growth inhibition. HMJ‐30‐induced apoptosis was mediated through both death‐receptor and mitochondria‐dependent apoptotic pathways in U‐2 OS cells. HMJ‐30 induced early phosphorylation of p53^Ser18 was through the activation of ataxia telangiectasia mutated (ATM) in U‐2 OS cells. The cell growth inhibition by HMJ‐30 was substantially attenuated either by the pre‐incubation of U‐2 OS cells with N‐acetylcysteine (NAC, an antioxidant) and caffeine (an ATM kinase inhibitor) or by p53 knockdown via RNAi. In conclusion, ROS dependent‐ATM/p53 signaling pathway is involved in HMJ‐30‐induced apoptosis in U‐2 OS cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1448–1456, 2011     

PI3K-AKT-mTOR signaling in prostate cancer progression and androgen deprivation therapy resistance

Prostate cancer （PCa） is the second most common malignancy among men in the world. Castration-resistant prostate cancer （CRPC） is the lethal form of the disease, which develops upon resistance to first line androgen deprivation therapy （ADT）. Emerging evidence demonstrates a key role for the PI3K-AKT-mTOR signaling axis in the development and maintenance of CRPC. This pathway, which is deregulated in the majority of advanced PCas, serves as a critical nexus for the integration of growth signals with downstream cellular processes such as protein synthesis, proliferation, survival, metabolism and differentiation, thus providing mechanisms for cancer cells to overcome the stress associated with androgen deprivation. Furthermore, preclinical studies have elucidated a direct connection between the PI3K-AKT-mTOR and androgen receptor （AR） signaling axes, revealing a dynamic interplay between these pathways during the development of ADT resistance. Thus, there is a clear rationale for the continued clinical development of a number of novel inhibitors of the PI3K pathway, which offer the potential of blocking CRPC growth and survival. In this review, we will explore the relevance of the PI3K-AKT-mTOR pathway in PCa progression and castration resistance in order to inform the clinical development of specific pathway inhibitors in advanced PCa. In addition, we will highlight current deficiencies in our clinical knowledge, most notably the need for biomarkers that can accurately predict for response to PI3K pathway inhibitors.     

Technology Insight: vaccine therapy for prostate cancer

The lack of effective therapies for advanced prostate cancer mandates continued development of alternative treatment strategies. Insights into the regulation of immune responses and the malignant process have facilitated the emergence of new immune-based strategies, currently under investigation in clinical trials. Like other forms of targeted therapy, cancer vaccines hold the promise of achieving cancer control without inducing overt toxicity. Many prostate cancer vaccines at different phases of development have been tested in clinical trials. Vaccination strategies under consideration include: immunization with defined antigenic preparations such as synthetic peptides, proteins or plasmid DNA; antigen-loaded dendritic cells; manipulated tumor cells; or with viral vectors engineered to express immunogenic genes. Although the underlying mechanisms of immunization may vary, all strategies share the common goal of eliciting immune responses against prostate tumor-associated antigens or of enhancing an otherwise weak antitumor response in the cancer patient. Unlocking the therapeutic potential of cancer vaccines will require a thorough understanding of cellular and molecular mechanisms that modulate the immune response. In this review, we provide an overview of vaccine-based strategies for prostate cancer therapy, discuss their mechanisms of action, and provide relevant clinical trial data.     

Stressing out over survival: glutamine as an apoptotic modulator

INTRODUCTION: The amino acid glutamine (GLN) has received considerable attention as a potential therapeutic adjuvant in critical illness and in improving postoperative clinical outcomes. Most studies on the role of GLN in cellular physiology have historically focused on its anabolic roles in specific cell types and its contribution to growth in cancer cells. However, an emerging body of work that examines the consequences of GLN deprivation on cellular survival and gene expression has constructed a new paradigm for this amino acid, namely, that limited extracellular GLN supplies modulate stress and apoptotic responses. METHODS: A survey of the scientific literature was conducted on GLN in cell survival signaling and apoptosis. Work from our laboratory in liver cancer cells also was included in this review. RESULTS: Most studies on this topic have used mammalian cell lines derived from the gut, immune system (including hybridomas), and various cancers. GLN limitation, even in the presence of an adequate glucose supply, impacts stress-related gene expression, differentially modulates receptor-mediated apoptosis, and directly elicits apoptosis through signaling mechanisms and caspase cascades that are specific to cell type. To date, GLN transporters, cellular hydration, glutaminyl-tRNA synthetase, ATP levels, mRNA stability, and glutathione economy have been variably implicated in GLN-dependent survival signaling. CONCLUSION: The cell type-specific mechanisms underlying the regulatory role of GLN in cell survival continue to unfold at a steady pace through in vitro studies. These results have collectively provided testable hypotheses for further in vivo studies into their physiological relevance during GLN "nutritional pharmacology."     

Prognostic Significance of Epidermal Growth Factor Receptor Expression in Colon Cancer Patients Undergoing Curative Surgery

Background To investigate the role of epidermal growth factor receptor (EGFR) expression as a prognostic marker for prediction of cancer behavior and clinical outcomes in colon cancer patients undergoing potentially curative surgery. Methods EGFR determination using a commercially available immunohistochemistry kit was performed in tissues from 149 colon cancer patients receiving primary surgical treatment and in 25 normal colon mucosa specimens from noncancer patients. EGFR positivity was correlated in univariate and multivariate analyses with disease recurrence and survival. In addition, p27, p53, and vascular endothelial growth factor expression were assessed by immunohistochemistry in 104 patients and correlated with EGFR tumor expression and clinical outcome. Results EGFR expression was detected in approximately one third of colon cancer patients (53 of 149; 35.6%). In 126 curatively treated patients, EGFR expression was correlated with disease recurrence and worse survival in both univariate and multivariate analyses. In a multivariate model for predicting recurrence and survival, Dukes’ staging, p27, and EGFR expression were the only independent covariates. In particular, in Dukes’ A and B patients the 5-year survival probability was 96% for EGFR-negative and high p27 expression cases and was 30.7% for EGFR-positive and low p27 expression cases. Conclusions EGFR expression was an independent prognostic indicator of disease recurrence and poor survival in colon cancer patients undergoing curative surgery. In the context of novel therapeutic options such as molecularly targeted therapies, these findings suggest that anti-EGFR drugs could be evaluated in the adjuvant treatment of EGFR-positive colon cancer patients.     

The basic biochemistry and molecular events of hormone therapy

Data regarding the molecular response of prostate cancer to hormone therapy continue to emerge, identifying a complex network of autocrine and paracrine signaling events mediating the tumor response to androgen suppression. Emerging data provide insight into cellular pathways important in the apoptotic response to therapy, including the transforming growth factor-β, insulin-like growth factor-1, and vascular endothelial growth factor signaling axes. They also reveal mechanisms of direct antitumor cytotoxicity mediated by various hormonal agents and highlight the importance of developing antiandrogens capable of irreversibly inhibiting the androgen receptor. Accumulated data emphasize the presence of residual androgens and persistent activation of androgen receptor signaling in advanced prostate tumors despite castration. These factors suggest that a multitargeted treatment approach designed to ablate all contributions to the androgen receptor signaling axis within the prostate tumor microenvironment will be required in order for hormonal therapy to achieve optimal antitumor efficacy.     

Medical Management of newly diagnosed breast cancer in a BRCA1/2 mutation carrier

Germline BRCA1/2 mutations may be infrequent in unselected breast cancer population but are concentrated in those with triple‐negative breast cancer or high‐risk family history. Insight into the biology of BRCA mutation is now allowing a targeted therapeutic approach to these carriers with breast cancer. Functional BRCA genes play a critical role in DNA damage repair. Agents such as platinum salts and poly (ADP‐ribose) polymerase (PARP) inhibitors exploit this vulnerability of impaired DNA damage repair mechanism in BRCA mutant cancers to leverage therapeutic benefit. Research has demonstrated improved response rates to platinum salts in BRCA‐mutated compared with non‐BRCA‐mutated breast cancer, particularly in the metastatic setting. Additionally, clinical trials of single‐agent PARP inhibitors have shown encouraging response rates and progression‐free survival in patients with BRCA1/2‐mutated breast cancer. In this review, we summarize the medical management of BRCA‐associated breast cancer.     

Radiofrequency ablation combined with palliative surgery may prolong survival of patients with advanced cancer of the pancreas

Background and aim The aim of this study is to identify the benefit acquired by the use of radiofrequency ablation in parallel to palliative therapy in patients with advanced cancer of the pancreas. Materials and methods Data on 25 consecutive patients who underwent palliative therapy with or without radiofrequency ablation for unresectable pancreatic cancer were included in this retrospective review. Thirteen patients received palliative therapy alone, whereas 12 patients received palliative therapy plus radiofrequency ablation. Results Overall mean survival rate in patients receiving paliative therapy alone was 13 months and the maximum survival was 30 months. Where radiofrequency ablation was applied, mean survival was estimated at 33 months (p = 0.0048). Stage III and IV patients treated with palliative therapy alone have a mean survival of 15 and 10 months, respectively. All stage III patients receiving radiofrequency ablation are alive at present and maximum survival has reached 38 months (p = 0.0032), whereas stage IV patients who were treated with radiofrequency ablation have an estimated mean survival period of 14 months (p = 0.1095). Conclusion Radiofrequency ablation in parallel to palliative therapy seems to provide survival benefit especially for stage III patients with unresectable pancreatic cancer. Further studies should be conducted to determine the usefulness of radiofrequency ablation in the treatment of advanced pancreatic cancer.