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Submitted in partial fulfillment of the requirement for the award of the degree of Bachelor of Technology In Biotechnology (2006-2010)
Guide: Dr. Rinu Sharma Assistant Professor
Submitted by: Saumya Singh 040/BT/USS/06
University School of Biotechnology Guru Gobind Singh Indraprastha University Kashmere Gate, Delhi-110403
Guru Gobind Singh Indraprastha University Kashmere Gate, Delhi
This is to certify that the project report entitled “Expression Analysis Of Stem Cell Marker Bmi1
In Tumor And Sera Of Esophageal Cancer Patients.” submitted by Mrs. Saumya Singh,
enrolment number 040/BT/USS/06, is an authentic work carried out by her at the University School of Biotechnology, Guru Gobind Singh Indraprastha University, under my guidance. The matter embodied in this project work has not been submitted earlier for award of any degree or diploma to the best of my knowledge and belief.
Signature of the student
Signature of the Guide
Mrs. Saumya Singh 040/BT/06
Dr. Rinu Sharma Assistant Professor University School of Biotechnology
With immense pleasure I wish to express my sincere gratitude to my mentor Dr.Rinu Sharma for her kind supervision, constant guidance, motivation, encouragement, and helpful advice throughout the course of this study. Without her valued suggestion and support this project would not have been a success. It has indeed been an enriching scientific experience and I would like to thank her for providing me an opportunity to work under her guidance. I am also grateful to Prof. R.K. Gupta, for providing me the best resources for my work. I am also indebted to Prof. P.C. Sharma, Dr .N. Raghuram, Dr. Meenu Kapoor, Dr. K.K. Aggarwal, Dr. Promila Gupta, Dr. Monika Gandhi, , Dr. H.K Das, Dr. Ram Singh Purthy, Dr. Suresh Kumar and Dr. Shalini for their support and encouragement throughout the course of the past four years and also during the course of this project. I would like to express my sincere thanks to Dr. Ranju Ralhan, Dr. Anoop Sarai (Department of Gastroenteritis), Mr. Hassan and Mr. Muzaffar for their guidance and support without which this project would not have been possible. They guided me during the project and made my learning, understanding and working process easy. I am also thankful to all my seniors Mr. Rajesh Ghangal, Ms Priyanka Sharma, Mr. Santosh, Mr. Sumer, Mr. Anand, Mr. Ravi and Mr. Manish, Mr. Ankit, Mr. Lunmin, Ms. Meenakshi, Ms. Deepika and Ms. Garima for their help, guidance, and encouragement during the period of this work. I would also like to thank all my colleagues and friends for their moral support and help throughout this project. I am also grateful to the laboratory staff of the School of Biotechnology, Mr. Puran Singh, Mr. Harinder, Mr. Ramesh, Mr. Kailash, Mrs. Nidhi and Mr. Goswami for their invaluable help and cooperation. 3
Finally I would like to thank my parents and my husband Mr. Sunit Nag whose love and support were a constant motivating factor during the course of this study.
Date: Place: New Delhi
Saumya Singh 40/BT/USS/05
To analyze the expression of stem cell marker Bmi1 in tissue and sera samples of Human Esophageal Squamous Cell Carcinoma (ESCC) patients and evaluate its potential as a blood based marker for diagnosis.
brain and colon tumors (Ricci-Vitian and Lombardi. In India it is the second most common cancer in males and the fourth most common cancer in females. pancreatic cancer (Li .. both of which are inconvenient and painful for the patient. breast cancer (Al-Hajj and Wicha. prognosis of esophageal cancer remains poor with a 5 year survival rate of only 5-10%. Thus there is a need for a noninvasive technique for ESCC detection. 2005).Berry. Current inadequacy of clinicopathological parameters as prognosticators warrants a clearer understanding of the molecular alterations in predicting prognosis of the disease (Mathew et al. and prostate tumors (Collins . Improvements in the prognosis and surveillance await a more comprehensive understanding of the molecular alterations and clinicopathological characteristics as predictors of a parsimonious model in tumor control. The patient thus avoids undergoing such diagnosis until the later stages arrives and by the time patients come to clinics the symptoms become more aggressive hence leading to poor prognosis.. Heidt and BA Daler. which will help in early and convenient diagnosis. increasing evidence has revealed cancer stem cells existing in at least certain kinds of tumors: acute myelogenous leukemia (Bonnet and Dick. have been characterized by many phenotypic traits similar to undifferentiated embryonic cells. 2002). Despite intensive multi-modality therapy including surgery. Cancer cells. Hyde. malignancy of proliferation and metastasis seems to be maintained and driven by such cancer stem cells. 2003). radiotherapy and chemotherapy. 1997). 2007). head and neck squamous cell carcinoma (Prince and Sivanandan. There is great urgency to isolate and generate clear molecular profiles of cancer stem cells for targeting antitumor 6 . In recent years. Various biological factors have been proposed as prognostic indicators of esophageal cancer. The present diagnostic tools for detection of ESCC are barium swallow and endoscopy of the esophagus. indicating that stem cell markers may be expressed in tumors..Abstract Esophageal cancer is the third most common cancer of the digestive tract and the seventh leading cause of cancer-related deaths worldwide (Globocan 2008). The embryonic stem cell factors are essential for pluripotency and self-renewal of embryonic stem cells. 2007).. especially in poorly differentiated or undifferentiated tumors... According to these studies. 2007).
With microarray analysis and quantitative real-time RT-PCR it is ascertained that several important stem cell–related genes (including OCT-4. BMI-1.treatment... 2009) and no report about the presence of BMI1 in sera has been made. Thus the present study was designed to investigate Bmi1 expression in ESCC and matched distant normal tissues as well as sera of esophageal cancer patients in order to evaluate its potential as non-invasive diagnostic marker for ESCCs... TTK.. 2005).. Bmi1 gene amplification is observed mainly in mantle cell lymphomas (Beà et al. 2004). non-small cell lung cancer (Vonlanthen et al. PTGS2. to date. SOX-2.... 1993) Several lines of evidence imply that Bmi1 plays an important role in the regulation of cell proliferation and senescence and is required for maintenance of adult hematopoietic and neural stem cells (Molofsky et al. and recent serial studies have shown that Bmi1 is over expressed in many somatic solid tumors such as colon carcinoma (Kim et al. and RBM15) were upregulated in cancer cells. 2001). and three Wnt and two Notch signal pathway related genes (such as FZD10. 7 . It was initially identified as an oncogene cooperating with c-Myc in the generation of lymphomas in double transgenic mice (Haupt et al. two ATPbinding cassette (ABC) transporter genes (ABCG2 and ABCA5). 2007) cell carcinoma and gastric carcinoma (Liu et al. and ZFX). breast cancer (Guo et al. 2005) tissues make it plausible that Bmi1 may play an important role in the initiation and development of ESCC. ( Dingzhi et al. 2007). The up-regulation of c-Myc and the down-regulation of p53 and p16 in ESCC (Kuwano et al.. 2009) Bmi1 is a member of the polycomb group (PcG) and a component of the polycomb repressive complex 1. 2008) and it may be of diagnostic and prognostic relevance. However. 2001). KLF5. head and neck squamous (Prince et al. only one report about the role of Bmi1 in ESCC has been made (Xiao-Ting et al.
3.2. Stem Cells 1. Review of literature 1.6 Implications for cancer treatment 8 .4 Evidence for cancer stem cells 184.108.40.206.1 Stem Cell Division 1.3.1 Types of Esophageal Cancer 1.2Cancer stem cells 1.6 Prognosis 1.3.3 Origin of Cancer Stem Cells 1.3 Signs and symptoms 1.CONTENTS 220.127.116.11 Pathophysiology and natural history 1.2 Esophageal cancer 1.5 Treatment 1.1Cancer 18.104.22.168.3.4 Diagnosis 1.5 Importance in cancer treatment 1.
22.214.171.124.2 Plastic and glassware 126.96.36.199.7 Challenges 1.1 RNA Isolation 3.1.8 Cancer stem cell markers 1.2RNA Isolation using RNeasy Mini Kit ( Qiagen) 9 . Materials and Methods 3.4 METHODS 3. Circulating mRNA 188.8.131.52.1 Materials 3.3 Primers 3.1TRI reagent protocol 184.108.40.206.2 TISSUE AND BLOOD SPECIMENS 3.5 Enzymes and PCR products 3.3 CELL LINES 3.1 General Chemicals 3. BMI-1 1.4. Objective of current study 3.4 Pipettes 3.
3 Denaturing Gel Electrophoresis of RNA 3. References Appendix Media Preparation Buffers 7.1.6 Agarose Gel Electrophoresis 4.4. Abbreviations 10 .5 Polymerase Chain Reaction 3.3 Isolation of total RNA from Sera Samples 3.2 RNA Quantification 220.127.116.11 Preparation of cDNA from reverse transcription reaction 3. Discussion 6. RESULTS 4.1 RNA Isolation 18.104.22.168.3 Reverse Transcription-PCR Analysis of stem cell related gene in ESCCs 5.2 Reverse Transcription-PCR Analysis of house-keeping Gene β actin 4.3.
lung. cervix. stomach. Review of literature 1. stomach (803 000 deaths). colorectal. 11 .3 million deaths/year). lung. ovary.breast.1Cancer Cancer affects people at all ages with the risk for most types increasing with age. uteri. thyroid. liver and esophagus Among women .6 million). More than 70% of all cancer deaths occurred in low. colorectal (639 000 deaths). stomach. The most frequent types of cancer worldwide (in order of the number of global deaths) are: • • Among men . The main types of cancer leading to overall cancer mortality each year are lung (1. corpus uteri. liver (610 000 deaths) and breast (519 000 deaths). colorectal.1.and middle-income countries. Cancer caused about 13% of all human deaths in 2007 (7. non-Hodgkin lymphoma and esophagus. with an estimated 12 million deaths in 2030. Deaths from cancer worldwide are projected to continue rising. leukemia. prostate.
The main type in these countries is squamous cell carcinoma. These figures take into account all patients with esophageal cancer. treatment has improved and survival rates are getting better.. no matter what stage they were in at diagnosis. depending on the type of cells that are malignant.. Prognosis depends on the extent of the disease and other medical problems. World Health Organization and GLOBOCAN 2008) 1. In some cases chemo. ( Chattopadhyay et al. 2009) 12 . Esophageal tumors usually lead to dysphagia (difficulty swallowing).and radiotherapy can render these larger tumors operable. 18% of white patients and 11% of African-American patients survive at least 5 years after diagnosis. Now. These cancers usually occur in the upper and middle part of the esophagus. Adenocarcinomas usually develop in the glandular tissue in the lower part of the esophagus. squamous cell carcinoma and adenocarcinoma. but is fairly poor. 2007) Although many people with esophageal cancer will go on to die from this disease. Iceland. Larger tumors tend not to be operable and hence are treated with palliative care. appear to have a higher incidence. their growth can still be delayed with chemotherapy. The treatment is similar for both types of esophageal cancer (Esophageal cancer. During the early 1960s. Mount Sinai Hospital). (Source: American Cancer Society. Iran. pain and other symptoms. only 4% of all white patients and 1% of all African-American patients survived at least 5 years after diagnosis.2 ESOPHAGEAL CANCER Cancer of esophagus (also called esophageal cancer) is divided into two major types. India and Japan. Squamous cell carcinomas arise in squamous cells that line the esophagus.(Source: WHO. radiotherapy or a combination of the two. Survival rates for early stage disease are higher.2003) Esophageal cancer is a relatively rare form of cancer. The highest incidence of this cancer in India has been reported from Assam in the North-east region where it is the second leading cancer in men and third leading in women. as well as the region around the Caspian Sea ( World cancer report 2003). (Enzinger and Mayer. but some world areas have a markedly higher incidence than others: China.. Small and localized tumors are treated surgically with curative intent. and are diagnosed with biopsy. as well as the United Kingdom.
diverticuli) that cause chronic irritation and inflammation of the esophageal mucosa may also increase the incidence of SCC. It is believed that the infection results in 13 . Source: everyday health 1. and esophageal webs—has been associated with this cancer. and this type of esophageal cancer may affect any part of the lining of the esophagus.2 Pathophysiology and natural history Smoking and heavy alcohol intakes are important risk factors for the development of SCC. and certain esophageal disorders (e.g. Smoking has a synergistic effect with heavy alcohol consumption. There are few genetic factors that have been identified as being important in the development of esophageal SCC.2.1 Types of Esophageal Cancer: The types of esophageal cancer are: Squamous Cell: Squamous cell esophageal cancer. Human papillomavirus has received the most attention. iron deficiency anemia. although it is becoming increasingly rare in the developed world as overall nutrition improves. and heavy exposure to both increases the risk of SCC by a factor of more than 100. the portion closest to the stomach. Vast majority of esophageal cancer develops in this inner lining.1. Infectious agents have also been implicated in the pathogenesis of esophageal SCC. or squamous cell carcinoma (SCC). Squamous cells make up the outermost layer of the esophageal lining. It develops in the lower part of the esophagus. One exception is tylosis. a rare autosomal dominant syndrome associated with hyperkeratosis of the palms and soles and a high rate of esophageal SCC.. Dietary and environmental factors. Adenocarcinoma: Adenocarcinoma (AC) is now the most common form of esophageal cancer in the United States.2. achalasia. Plummer-Vinson syndrome—the triad of dysphagia. is the more frequently diagnosed type of esophageal cancer around the world.
and tumor-related anorexia. Dysphagia. or vertebral bodies. Although this mucosal change appears to be a favorable adaptation to chronic reflux—columnar epithelium appears to be more resistant to reflux-induced injury than the native squamous cells—this specialized intestinal metaplasia may become dysplastic and ultimately malignant. liquids. Weight loss is common and correlates with dysphagia. such as the pleura. if present. Most esophageal ACs are believed to arise from Barrett's esophagus. dietary changes. The risk factors for AC of the esophagus are different.3 Signs and symptoms The clinical presentation of patients with esophageal cancer can be attributed to the direct effects of the local tumor. such as obesity or medications that lower the lower esophageal sphincter tone. with severe. Source: Esophageal cancer. Weight loss is noted in more than 70% of patients and. carries a worse prognosis. usually develops in response to dense solid food. may result in an increased risk for esophageal AC. Factors that increase the risk for gastroesophageal reflux. Thomas Rice. the most common manifesting symptom. Ahmed Absi. Adelstein. 1. finally. or paraneoplastic syndromes. regional or distant complications of the disease. Chronic gastroesophageal reflux is the most important.loss of function of the tumor suppressor genes p53 and Rb. David J. and progresses gradually to interfere with the intake of softer foods and. AC and SCC have similar clinical manifestations. mediastinum. The importance of this mechanism is not well established. It can be related to swallowing itself (odynophagia) or to the local extension of the tumor into adjacent structures. a condition in which an abnormal columnar epithelium replaces the stratified squamous epithelium that normally lines the distal esophagus. particularly in patients with advanced local disease. This can sometimes be accompanied by vomiting or regurgitation of saliva or food uncontaminated by gastric secretions. or both. Other manifesting signs and symptoms reflect 14 . which reflect the extent of local esophageal involvement. disable tumor suppressor genes. Chronic gastroesophageal reflux disease is associated with Barrett's metaplasia (Barrett's esophagus).2. long-standing reflux symptoms increasing the risk of cancer by a factor of 40. with genetic alterations that activate proto-oncogenes. Pain is frequent and can occur in the absence of dysphagia.
such as cough or fever from a respiratory tract fistula. bone. including supraclavicular lymphadenopathy. and central nervous system Hypercalcemia system. particularly in the case of AC. May 2009) Manifesting Symptoms of Esophageal Cancer Symptoms Caused by Local Tumor Effects • • • • • Dysphagia Cough and regurgitation Odynophagia Weight loss Upper gastrointestinal bleeding Respiratory fistula Hoarseness from recurrent laryngeal nerve invasion Hiccups from phrenic nerve invasion Pain caused by local spread Metastatic disease to the lungs. hepatosplenomegaly. In the absence of bone metastases. upper or lower gastrointestinal bleeding. hoarseness from recurrent laryngeal nerve involvement.. The physical examination is often unremarkable. and central nervous 15 . and hiccups from phrenic nerve involvement. and pleural effusion. liver.complications from disease spread. it is most common in patients with SCC and is believed to be caused by the production of a parathyroid hormone–related protein. ( Piero Marco Fisichella. but should be directed toward finding evidence of metastatic disease. can also be found at the initial clinical presentation. Hypercalcemia is the most common paraneoplastic syndrome. liver. Symptoms Related to Invasion of Surrounding Structures • • • • Symptoms Related to Distant Disease • • Symptoms related to distant metastasis in the lungs. eMedicine specialities.
Aging alone causes mild esophageal motility abnormalities. Two historical reviews of the outcomes after esophageal cancer treatment. have demonstrated this clearly.. 1999) The overall 5-year survival rate was 4% after surgical resection. and mandates the need for an immediate evaluation to define its exact cause and initiate appropriate therapy. More than 50% of patients have unresectable or metastatic disease at the time of presentation. Any radiation therapy portal encompassing the esophagus will also include other vital structures. After establishing a diagnosis of esophageal cancer. Dysphagia in older adults should not be attributed to normal aging. The frequent medical co morbidities and high incidence of second malignancies in these patients also affect the overall treatment success.2. Although modern radiation techniques have fewer adverse side effects. and lungs. is challenging because of the anatomic location of the esophagus. endoscopic ultrasonography and. major airways. high-dose definitive radiation therapy. if appropriate.1. 1.5 Treatment Historically. as an alternative to surgical resection. Combining these techniques yields an overall diagnostic accuracy of 98%. published in 1980. adequate staging is required. Furthermore. Optimal clinical staging for this disease should include at least computed tomography (CT) scanning of the chest and abdomen. 16 . but these are rarely symptomatic. because staging is the most important step in choosing appropriate therapy. toxicity is still common with the radiation doses required. Endoscopy will allow the direct visualization of any tumor mass and histologic confirmation with a biopsy or brush cytology. such as major blood vessels. the heart. esophageal cancer has carried a dismal prognosis. Evaluation of dysphagia starts with a barium swallow examination or an upper endoscopy. (Lagergren et al. For the others.2. a positron emission tomography (PET) scan. survival is closely related to the stage of the disease. This has been attributed to the late presentation of patients with this disease and the technical difficulty of an adequate surgical resection in the presence of advanced local and regional involvement.4 Diagnosis Dysphagia is an alarming symptom.
. A cisplatin-based regimen is often used. single-modality radiation therapy should. Surgical results have improved significantly over recent years... The transhiatal approach requires a laparotomy. 2007) In patients with tumors of the gastroesophageal junction and significant gastric involvement. using a transthoracic or transhiatal approach. be considered a palliative intervention in patients whose underlying medical co morbidities preclude surgical resection or aggressive multimodality treatment. 2006) For more advanced disease. Radiation therapy has been used in the past as a single-modality approach with curative intent. Multiple surgical series from major medical centers now report that patients undergoing surgery alone have median survival rates between 13 and 19 months. This approach saves the patient the cardiopulmonary complications of a thoracotomy. 2003) Although these numbers are certainly more promising. however. Preoperative chemotherapy has been studied in several randomized 17 . but no prospective clinical trials have demonstrated superiority of this procedure over a thoracoabdominal approach (Merry et al. Given the propensity for submucosal skip lesions in the esophagus. or both. especially when we keep in mind that much of this improvement is the result of better clinical staging and better patient selection. postoperatively. radiation has had little impact on long-term survival (Layke and Lopez. this usually requires a subtotal esophagectomy. in general.with an unacceptable surgical mortality rate of almost 30%. Multimodality treatment approaches have evolved over recent years in response to the frequent locoregional and distant recurrences identified after surgery or radiation therapy alone. However. The overall 5-year survival rate was only 6% after radiation therapy. 2-year survival rates between 35% and 42%. a total gastrectomy and Roux-en-Y esophagojejunostomy may be required. except for those with very early-stage disease. Several different combinations and sequences of treatment modalities have been tried. they can hardly be characterized as a medical success story. and esophagogastric anastomosis in the neck. Chemotherapy has been given preoperatively. blunt dissection of the thoracic esophagus. and 5-year survival rates of 15% to 24% ( Green et al. The goal of oncologic surgery for this disease is the resection of the primary tumor and draining lymph nodes. with mixed results.
that 25% to 30% of patients treated in this fashion achieved a pathologically complete response after concurrent chemoradiation therapy. this trimodality approach produces considerable toxicity. however. These studies have demonstrated that induction chemotherapy can produce up to a 50% clinical response rate but less than a 10% pathologic complete response rate. photodynamic therapy) (Brooks et al.. the results of these studies have been mixed. endoscopic stenting. (Layke 2006) How this approach can be integrated with surgery has remained unclear. For obvious reasons. in many major referral centers in the United States. Patients with metastatic disease are treated with palliative intent. trimodality therapy is used for suitable patients who have at least T3 lesions. 2009) Current efforts are directed toward developing targeted therapies that may prove more active in esophageal cancer. (Islami. However. any nodal involvement with esophageal cancer. It is of note. This can be achieved in several ways. 18 .g. Herskovic and colleagues (Layke. which produced no long-term survivors. More intensive multimodality approaches have attempted to exploit the radiosensitizing properties of chemotherapy by using concurrent cisplatin-based chemotherapy and radiation as definitive treatment or as a preoperative adjuvant. A clear survival benefit for the combined approach was identified.clinical trials that compared surgery alone with chemotherapy followed by surgery.. endoscopic dilation. with a 5-year survival rate of 25% compared with radiation therapy alone. Lopez.. the results have been conflicting. including palliative radiation. 2006) reported a phase III prospective randomized trial that compared chemotherapy given concurrently with radiation with radiation therapy alone. however. 2009). Unfortunately. and that the 2-year survival rate after subsequent surgery is approximately 35%. endoscopic laser therapy. with no consistent survival advantage identified for one approach or another.. et al. or both. Surgery was not a planned part of disease management in this trial. with some series reporting treatmentrelated mortality in excess of 12% (Whiteman. and no clear survival advantage has been identified with the induction regimens. In 1992. 2009). Palliation should first be directed toward relief of dysphagia and esophageal obstruction. Currently. Three small phase 3 trials have randomized patients to surgery alone or to preoperative concurrent chemoradiation followed by surgery. or other light-based therapy (e..
Those with cancer restricted entirely to the esophageal mucosa have about an 80% 5YSR. skin or intestinal tissues. There are two main types of stem cells: embryonic stem cells and adult stem cells. In a tissue such as the esophageal epithelium that has a single differentiation pathway. They serve as a repair system for the body.3. the prognosis of esophageal cancer is quite poor. stem cells and progenitor cells act as a repair system for the body. Source: NIH: National Institutes of Health Stem cells are cells found in all multi cellular organisms. Stem Cells Stem cells are cells with the potential to develop into many different types of cells in the body. Individualized prognosis depends largely on stage. Transit amplifying cells undergo several rounds of division. In adult organisms. replenishing specialized cells.3. such as blood. 1. tissue renewal is performed by the transit amplifying cell population. Differentiation is associated with a reduction in proliferative capacity.1 Stem Cell Division Stem Cell division serves two purposes. and ultimately the differentiated progeny are lost. These stem cells are present in limited number residing in the generative layer of the tissue. First it replenishes the stem cell compartment and secondly it generates transit amplifying cells. Patients with distant metastases (who are not candidates for curative surgery) have a less than 3% 5YSR. because so many patients present with advanced disease: The overall five-year survival rate (5YSR) is less than 5%. but also maintain the normal turnover of regenerative organs. but submucosal involvement brings this down to less than 50%. and their progeny eventually execute the differentiation pathways characteristic of the tissue where the stem cells reside. 19 . They are characterized by the ability to renew themselves through mitotic cell division and differentiate into a diverse range of specialized cell types. 1. Extension into the muscularis propria (muscular layer of the esophagus) has meant a 20% 5YSR and extension to the structures adjacent to the esophagus results in a 7% 5YSR.Prognosis In general.
• The difference in function between stem cells and transit amplifying cells is likely to be reflected in different levels of expression of functionally relevant molecules.( Tannishtha Reya et al. But stem cells have a higher proliferation capacity as the progeny of transit cells undergo terminal differentiation after several rounds of division. ( Seery 2002) 1. 2001) 20 . • The stem cells should proliferate less frequently in vivo relative to the transit amplifying population in the steady state.3. similar signalling pathways may regulate selfrenewal in stem cells and cancer cells. and cancer cells may include 'cancer stem cells' — rare cells with indefinite potential for self-renewal that drive tumorigenesis..2CANCER STEM CELLS Unequivocal proof that stem cells exist in the hematopoietic system has given way to the prospective isolation of several tissue-specific stem and progenitor cells. and the beginnings of their utility in regenerative medicine. striking parallels can be found between stem cells and cancer cells: tumors may often originate from the transformation of normal stem cells. the initial delineation of their properties and expressed genetic programmes.Several predictions have been made by scientists that allow identification and isolation of putative human stem cells. Perhaps the most important and useful property of stem cells is that of self-renewal. • Stem cells do not display differentiation markers on their surface as they do not generally initiate a program of differentiation and hence are phenotypically primitive. Therefore such molecules can be used in the identification and isolation of the two cell types. Through this property.
induced by physical or chemical mutagens. are substrates that can be fixed if they 21 .FigureI: A diagrammatic heuristic scheme to depict the postulated mechanisms of the initiation and promotion phase of carcinogenesis. DNA lesions.
cancer stem cells remain viable. Promotion includes those conditions (i. the existence of cancer stem cells has been proven in acute and chronic myeloid leukemia. in brain tumors. might allow a given cell to have autonomous. The buildup of initiated cells allows them to “resist” the antimitotic influence of neighboring noninitiated cells.. invasive properties of a malignant cell. Although the concept that cancers arise from “stem cells” or “germ cells” was first proposed about 150 years ago. and express typical markers of stem cells.e. Probably both mechanisms are involved in the origin of cancer stem cells. initiated cell can escape the nonproliferative state. exogenous promoters) in which a pluripotent. Cancer stem cells have the ability of self-renewal and proliferation.” Two important related concepts of this hypothesis are that (a) tumors originate in either tissue stem cells or their immediate progeny through dysregulation of the normally tightly regulated process of self-renewal. but surviving. Although standard chemotherapy kills most cells in a tumor. As a result of this. Further characterization of cancer stem cells is needed in order to find ways to destroy them. in lung cancer and gastrointestinal tumors. together with a second mutation. Despite the small number of such cells. Cancer stem cell model is also consistent with some clinical observations. (From Trosko and Tai). This. they might be the cause of tumor recurrence. wounding. Dysregulation of stem cell self-renewal is a likely requirement for the development of cancer. sometimes many years after the "successful" treatment of primary tumor. (b) tumors contain a cellular 22 . are resistant to drugs. in breast cancer. it is only recently that advances in stem cell biology have given new impetus to the “cancer stem cell hypothesis. It is not clear whether cancer stem cells originate from normal stem cells in consequence of genetic and epigenetic changes and/or by redifferentiation from somatic tumor cells to the stem-like cells. Growth of metastases in distinct areas of body and their cellular heterogeneity might be consequence of cancer stem cell differentiation and/or dedifferentiation and asymmetric division of cancer stem cells. Isolation and identification of cancer stem cells in human tumors and in tumor cell lines has been successful. To date. which might contribute significantly to the therapeutic management of malignant tumors. cytotoxicity.are not removed in an error-free manner prior to DNA replication.
Stem cells by their long-lived nature are subject to the accumulation of multiple mutations that are required for carcinogenesis. Recent experimental evidence in a variety of tumors has lent strong support to the cancer stem cell hypothesis that represents a paradigm shift in our understanding of carcinogenesis and tumor cell biology.3 Origin of Cancer Stem Cells Two important factors need to be considered while recognizing the origin of cancer stem cells. 1973) and 2) a stem cell needs to overcome any genetic constraints on both self-renewal and proliferation capabilities (Morrison et al. For example.subcomponent that retains key stem cell properties. 2004). 2002). cancer stem cells should be derived from either the mutations in normal stem cells or from the progenitor cells that have acquired the ability of self-renewal due to multiple oncogenic mutations (Al-Hajj et al. Therefore. 1999) 1. Furthermore. This hypothesis has fundamental implications for cancer risk assessment. women exposed to atomic bomb radiation in Hiroshima and Nagasaki developed breast cancer approximately 20 to 30 years after exposure (Little et al. It is unlikely that all the mutations could occur alone in the lifespan of a progenitor/mature cell.. and prevention.. which drives tumorigenesis. early detection. (Cancer Res 2006 1883-90) Evidence supporting the cancer stem cell hypothesis has gained impetus due to recent advances in stem cell biology and the development of new animal models to measure self-renewal and more directly test the validity of this hypothesis.3.. and differentiation albeit aberrant that contributes to cellular heterogeneity. 23 . prognostication. the current development of cancer therapeutics based on tumor regression may have produced agents that kill differentiated tumor cells while sparing the rare cancer stem cell population. These properties include selfrenewal. 1) a number of mutations are required for a cell to be cancerous (Knudson et al. The development of more effective cancer therapies may thus require targeting this important cell population.. The concept that cancers arise from the transformation of stem cells is appealing for several reasons.
2007) Ovary (Zhang. (3) rate of cell division of initiated cells. 1997) The existence of leukaemic stem cells prompted further research into other types of cancer. (Bonnet and Dick. This implies that the cell that produced them had the capacity to generate multiple cell types.. including cancers of the: Brain (Singh SK Cancer research 2003) Breast (Al-Hajj et al. the study of the tissue structure of tumors. mutation). Heterogeneity is commonly retained by tumor metastases.. a classical hallmark of stem cells. In other words.. Lang et al.4 Evidence for cancer stem cells Evidence comes from histology. (4) rate of the molecular event leading to initiation (i. Cancer research2007) Prostate ( Maitland et al 2008.) 1. Cancer research2008) Pancreas (Li C. CSCs have recently been identified in several solid tumors. it possessed multidifferentiative potential.e..3. 2003) Colon (O'Brien et al. but not of terminal differentiation of the initiated cell. and (5) rate at which the second event occurs within an initiated cell (from Trosko et al... (2) rate of death.2009) 24 . Heidt. Many tumors are very heterogeneous and contain multiple cell types native to the host organ.Figure II: The initiation/promotion/progression model of carcinogenesis showing (1) rate of terminal differentiation and death of stem cell.
6 Implications for cancer treatment The existence of CSCs has several implications in terms of future cancer treatment and therapies. these cancer stem cells may emigrate to distal sites from the primary tumor and cause metastasis via blood stream (Jordan et al.3.1. 2002). • • First. DNA repair proteins and they also have a slow rate 25 . These include disease identification. most of the primary tumor cells may be destroyed but the cancer stem cells are generally not eradicated due to their drug pumping property and also they have been shown to have different sensitivity to different chemotherapeutic agents such as TICs in leukemia are less sensitive to daunorubicin (Costello et al. 2000) and cytarabine (Guzman et al.. For tumors in which the cancer stem cells play role. selective drug targets... 1. Hence the majority of treatments target the rapidly dividing cells that represent the bulk of the tumor. Normal somatic stem cells are naturally resistant to chemotherapeutic agents. three possibilities exist owing to their properties as discussed above. identification of the cancer stem cells may allow the development of treatment modalities that target the cancer stem cells rather than rapidly dividing cells in the cancer. Second. they become refractory cancer stem cells and may lead to the relapse of the disease.3. Theoretically. 2006). and development of new intervention strategies..they have various pumps (such as MDR) that pump out drugs. the cancer treatment is targeted at the proliferation potential of the cancer cells and their ability to metastasize. • Third..5 Importance in cancer treatment At present. This may explain the failure of treatments to eradicate the disease or the recurrence of the cancer (Reya et al. these effects are transient and usually do not improve patient's survival outcomes (Stockler et al. Although current therapies may reduce the size of the tumor.So. This may cure the cancer as the remaining cells in the cancer growth have limited proliferative capability. during chemotherapy. 2001). 2000). the mutation of normal stem cells or progenitor cells into cancer stem cells can lead to the development of the primary tumor. prevention of metastasis.
Notch and Wnt that are crucial for normal stem cell regulation. have been shown to be deregulated in the process of carcinogenesis (Galderisi et al. In 2009. Salinomycin. as well as preventing the tumor from metastasizing. 2005). a commonly used chemotherapeutic agent (Deschamps. By selectively targeting CSCs. 1. 2006). (Gallus 2003) A number of studies have investigated the possibility of identifying specific markers that may distinguish CSCs from the bulk of the tumor (as well as from normal stem cells) ( Syrjänen 2002) Proteomic and genomic signatures of tumors are also being investigated . 26 . which traditionally target the rapidly dividing cells. These make the cancer stem cells resistant to the toxicity of the anti-cancer drugs. These deregulated signalling pathways and gene expressions may have impact on response to cancer therapy. CSCs that have mutated from normal stem cells may also express proteins that would increase their resistance towards chemotherapeutic agents.. The hypothesis suggests that upon CSC elimination..of cell turnover (chemotherapeutic agents naturally target rapidly replicating cells). polycomb gene Bmi1 (Park et al. that selectively reduces the proportion of breast CSCs in mice by more than 100-fold relative to Paclitaxel. scientists identified one compound..7 Challenges Although the idea of the therapies focused on the cancer stem cells may look exciting. targeting the cancer stem cells may not be easy.3. cancer would regress due to differentiation and/or cell death What fraction of tumor cells are CSCs and therefore need to be eliminated is not clear yet. non-resectable tumors. causing relapse. Various reasons are: • The cancer stem cells are relatively quiescent compared to other cancer cells and do not appear to have the hyper-proliferation signals activated such as tyrosine kinase. These surviving CSCs then repopulate the tumor. the tumor suppressor gene PTEN (Groszer et al. 2003) and the signal transduction pathways such as the Sonic Hedgehog (Shh). 2001). • In addition.. it would be possible to treat patients with aggressive.
The stem cells are immortal.• Also there is a need to identify the markers that are specific for the cancer stem cells compared to normal stem cells such as hematopoietic stem cells express Thy-1 and c-kit whereas leukaemic stem cells express IL-3 (interleukin-3) receptor α-chain (Blair et al. 1. this makes them hard to find and even more difficult to target.(Burger et al. 2000). 2009). (Li et al. 1997.. 2009). there is a need to identify and validate different markers to isolate and characterize CSCs. CD200. 2009). Almost all of these ALDH high cells also express Sca-1 and a third of them express high levels of this antigen. 2009). 2009).. With the increasing evidence for the "cancer stem cell (CSC) hypothesis".. such as the ability of selfrenewal. particularly ALDH1in the circulating tumor cells of metastatic breast cancer patients (Aktas et al. CD24. Pancreatic cancer stem cells also demonstrate upregulation of molecules important in developmental signaling pathways. ABCG2 and nestin). a recently discovered intestinal stem cell marker. Normal stem cells in the adult organism are responsible for tissue renewal and repair of aged or damaged tissue.. Oct 3/4.. Lgr5 (leucine-rich-repeat-containing Gprotein-coupled receptor 5). CD133 found on colon cancer stem cells (Li et al. is expressed in 27 . differentiation and proliferation and demonstrate higher ability of colony-forming ex vivo and tumorigenesis in vivo (Sun et al. Some of the potential cancer stem-cell marker identified are the enzyme aldehyde dehydrogenase activity. High levels of aldehyde dehydrogenase (ALDH) activity are also present in a subset of prostate epithelial cells that co-express a number of antigens found on stem/progenitor cells of other origins (CD9. and rather resistant to action of drugs. Bcl-2. including sonic hedgehog and the polycomb gene family member Bmi-1. Blair et al..3. prominin. p75NTR positive cells carry some properties of cancer stem cells.8 CANCER STEM CELL MARKERS There is increasing evidence supporting the cancer stem cell hypothesis. They are able to differentiate and form specific types of tissue due to the influence of microenvironmental and some other factors.. Since only a small percentage of cells in a tumor are cancer stem cells. A substantial characteristic of stem cells is their ability for self-renewal without loss of proliferation capacity with each cell division. or ALDH.
breast. Bmi1 is necessary for efficient selfrenewing cell divisions of adult hematopoietic stem cells as well as adult peripheral and central nervous system neural stem cells and is a downstream target in the Hedgehog (Hh) pathway.. (Yadav et al.premalignant lesions including Barrett's esophagus (BE) and cancers including colon cancer. such as bladder. it is possible that it regulates the self28 .4.. 2010) Aberrant expression of BMI1 has been linked to cancer stem cell phenotype and oncogenesis. BMI-1 BMI-1 polycomb ring finger oncogene. (NCBI sequence viewer). Given that phenotypic changes in Bmi1 knockout mice are numerous and that Bmi1 has very broad tissue distribution. it is less important for the generation of differentiated progeny. skeletal patterning.. 1. (Becker et al. This is why. In Homo sapiens. cancer and development. It's amplification and overexpression is especially pronounced in mantle cell lymphomas (Shakhova. the cell cycle inhibitor genes and hence act as the oncogene rendering abnormal proliferative ability. its overexpression has been found in several human malignancies including breast cancer. neurological functions. and hepatocellular carcinoma. 2009). 1993). (Itahana et al. In particular. The Accession Number for BMI-1 gene is NM_005180. 2003) The polycomb group (PcG) proteins form chromatin-modifying complexes that are commonly deregulated in cancer. prostate. Bmi1 knockout in mice results in defects in hematopoiesis. 2005) BMI-1 has been reported to regulate p16 and p19. at present not much is known about the functional domains of BMI1 oncoprotein. the gene is located on 10th chromosome. skin. is a protein which in humans is encoded by the BMI1 gene (Alkema et al. The mRNA transcribed from this gene is linear and 3251 bp in length. Bmi1 has a RING finger at the N-terminus and a central helix-turn-helix domain.. The PcG protein BMI-I is overexpressed by various tumors and thus may contribute to malignant transformation.. ovarian cancer.5 (GI: 39725706). and development of the cerebellum. also known as BMI-1. Bmi1 seems to play an important role in several types of cancer. ovarian. Despite its established role in stem cell maintenance. However. colorectal as well as hematological malignancies.
8%) such patients showed Bmi-1 positive expression. A blood-based assay. These signaling pathways control the cell cycle. potentially. Bmi-1 has also been suggested to extend the replicative life span of human fibroblasts by suppressing the p16-dependent senescence pathway. 2009) 1. 2002) In approximately 11% of cases of mantle cell lymphoma. implicating this gene in this invariably lethal form of lymphoma. 2004) Bmi1 is also thought to inhibit ageing in neurons by suppressing p53 and the induction of a premature aging-like program characterized by reduced antioxidant defenses. the malignant cells have a three to even old amplification of Bmi1 DNA and express high levels of the protein. The patients with Bmi-1-positive cancers showed significantly poorer prognoses than those without. Circulating mRNA as potential diagnostic tools for cancer Evidence from the literature widely supports the efficacy of screening and prognosis for cancer in reducing mortality. represents a more accessible early detection tool for the identification of circulating tumor cells originating from a primary tumor site in the body (Sheng-Li Zhou and Li-Dong Wang. The role of Bmi-1 expression in esophageal cancer progression has also been analyzed whether it could be used to predict disease progression and prognosis in ESCC patients undergoing neoadjuvant chemoradiotherapy (CRT).. cellular senescence and cell death.. (Grinstein and Mahotka. 2010).. further elucidation thereof in stem cells might have implications in cancer research. 24 out of 78 (30.. ( Itahana et al.. These mRNAs may represent useful 29 . While the roles of the pathways associated with Rb and p53 in cancer are broadly established. It has been shown that Bmi-1 expression is associated with early relapse and poor prognosis in ESCC after CRT suggesting that aberrant Bmi-1 signal activation as well as Hh pathway activation may be indicative of emergence of more aggressive cancer progenitor cells and therapeutic target after CRT (Fujiwara et al. cell differentiation. 2009) BMI-1 interferes with the central cellular tumor suppressor pathways linked to retinoblastoma protein (Rb) and p53.renewal of other types of somatic stem cells (Park et al. stem cell biology and regenerative medicine.5.
To analyze the mRNA expression of stem cell related gene viz. following RNA extraction from peripheral blood samples 2. 2). 30 . To analyze the expression of circulatory mRNAs of this gene in the sera of Esophageal Carcinoma patients with the aim of analyzing their potential as non-invasive biomarkers for diagnosis/prognosis. OBJECTIVES Following objectives were considered to perform the study: 1).markers for early detection of circulating cancer cells by a simple. BMI-1 in Esophageal Squamous Cell Carcinoma and matched normal tissues using Reverse Transcription Polymerase Chain Reaction (RT-PCR). qualitative RT-PCR assay.
petridishes and other plasticwares were purchased from Tarsons (India) and laboratory glassware from Borosil (India). Materials 3.1. MATERIAL AND METHODS 3.1.1. microfuge tubes. (Gentech). Louis.S. USA . 3. Ethylene diammine tetrachloride. SISCO Research Laboratories.A.1 General Chemicals Rose Park Memorial Institute (RPMI) medium. KCl. U.3. 3. boric acid.1. Tris ethyl ammonium chloride. eppendorfs and pipette tips were purchased from Axygen (USA). Reagents used for cDNA preparation and the Pcr Reagents were obtained from Fermentas.3 Primers 31 . Na2HPO4 and KH2PO4 were obtained from Qualigens. ciprofloxacin antibiotics. St. Ethidium bromide and agarose was purchased from Sigma Chemical Co. NaCl. Fetal Bovine Serum (FBS) were obtained from GIBCO BRL.2 Plastic and glassware PCR tubes. MA.
3 CELL LINES 32 .5 Enzymes and reagents for PCR and cDNA preparation All the reagents and enzymes used in RT-PCR reactions viz. 3. hot tea and oil was prepared. 3.Custom synthesized primers for RT-PCR (Hexamers) and Gene specific PCR primers (Bmi-1 and βActin) were obtained from SBS Genetech Co.2 TISSUE AND BLOOD SPECIMENS Endoscopically resected tissue specimens from esophageal region were obtained from Department of Gastrointestinal surgery. alcohol. New Delhi. Specimens of normal esophageal tissues were obtained from cancer patients from an area distant to the site of the Esophageal lesions. Mumbai. All India Institute of Medical Sciences. Taq DNA Polymerase. Chilies. India. India 3. Ltd.1. Blood samples from patients with esophageal cancer were also collected. 3.4 Pipettes Single pipettes were obtained from Biohit. India. The serum was separated and stored in -80oC till use. gender. histopathological differentiation and food habits like consumption of tobacco (smoke and smokeless). RevertAid™ H Minus M-MuLV Reverse Transcriptase including the GeneRuler™ 100 bp DNA Ladder were obtained from Fermentas.1. Specimens were collected and were immediately snap frozen in liquid N2 and stored at -80oC till further use for RNA analysis. A predesigned performa for recording the clinical and pathological data including age.
• After centrifugation.4.1. 250 µl was mixed with 750 µl of Tri reagent. interphase and the colorless upper aqueous phase. The samples were covered tightly and shaken vigorously for 15 seconds.4 METHODS 3.000 g for 15 minutes at 4 oC.The Esophageal squamous cell carcinoma (SCC) cell line TE13 was used as positive control for Gene Expression studies.1TRI reagent protocol • Tissue specimen snap frozen in liquid N2 was minced using surgical blades or crushed in liquid N2 using motor pestle. 1993). the mixture separated into a lower red phenol-chloroform phase. • The homogenate was supplemented with 0.100 mg of tissue. Cells were grown in monolayer cultures in a humidified incubator (5% carbon-dioxide. Oral cancer cell line. U87 were also used in the study of Stem cell markers.2 ml chloroform per 1 ml of TRI Reagent. • The resulting mixture was stored at room temperature for 2-15 minutes and was centrifuged at 12. CCS4 and Glioma cell line. Then 1ml of Trireagent was added per 50 .. 3. The Cell lines were first washed with PBS solution to get rid of any residual media. • • In case of Serum samples.4.1 RNA Isolation 3. Then 1ml of Trireagent was flushed in the flask to detach the cells from the surface. 33 . • The homogenate in all the three cases was stored for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes. 95% air) at 37oC. TE13 cells were cultured in a 1:1 mixture of Rose Park Memorial Institute (RPMI) supplemented with 10% FBS. The cell line was developed from squamous cell carcinoma of esophageal origin (Nishihira et al.
15 minutes. RNA was dissolved in diethyl pyrocarbonate (DPEC) treated autoclaved water (20 µl) by passing the solution a few times through a pipette tip and incubating for 10-15 minutes at 65 oC.5 ml of isopropanol was used per 1 ml of TRI Reagent for the initial homogenization.500 g for 5 minutes at 4 oC. • The aqueous phase was transferred to a fresh tube and the interphase and organic phase was saved at -20 oC for subsequent isolation of DNA and proteins. 34 . • • Ethanol was removed and the RNA pellet was air-dried for 2 . • RNA was stored at -80 oC for long term storage.• RNA remained exclusively in the aqueous phase whereas DNA and proteins were in the interphase and organic phase. • RNA was precipitated and formed a gel-like or white pellet on the side and bottom of the tube.000 g for 10 minutes at 4 oC. • RNA was precipitated from the aqueous phase by mixing with isopropanol. At least 1 ml of 75% ethanol per 1 ml TRI Reagent was used for the initial homogenization. Samples were store at room temperature for 5-10 minutes and centrifuge at 12. • The supernatant was removed and the RNA pellet was washed (by vortexing) with 75% ethanol and subsequently centrifuged at 7. 0.
1. 1a. Harvest cells according to step 1a or 1b.Figure III: TRI REAGENT Protocol 3. affecting the conditions for binding of RNA to the RNeasy membrane. Completely aspirate the cell-culture medium.2RNA Isolation using RNeasy Mini Kit ( Qiagen) 1. 35 . Carefully remove all supernatant by aspiration. and proceed to step 2. Both effects may reduce RNA yield. Cells grown in cell-culture flasks should always be trypsinized.4. Cells grown in suspension (do not use more than 1 x 107 cells): Determine the number of cells. 1b. Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute the lysate. Cells grown in a monolayer (do not use more than 1 x 107 cells): Cells can be either lysed directly in the cell-culture vessel (up to 10 cm diameter) or trypsinized and collected as a cell pellet prior to lysis. and proceed immediately to step 2. Pellet the appropriate number of cells by centrifuging for 5 min at 300 x g in a centrifuge tube (not supplied). To lyse cells directly: Determine the number of cells.
affecting the conditions for binding of RNA to the RNeasy membrane. and add 0. Volumes of Buffer RLT for Lysing Pelleted Cells Number of Volume RLT ( µl) 350 600 of buffer pelleted cells <5 x 106 5 x 106 – 1 x 107 For direct lysis of cells grown in a monolayer. Both effects may reduce RNA yield. loosen the cell pellet thoroughly by flicking the tube. Disrupt the cells by adding Buffer RLT. and wash the cells with PBS. Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute the lysate. Pipet the lysate into a microcentrifuge tube. add medium (containing serum to inactivate the trypsin). Vortex or pipet to mix. Add the appropriate volume of Buffer RLT (see Table 5). For pelleted cells.Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute the lysate. Volumes of Buffer RLT for Direct Cell Lysis 36 . transfer the cells to an RNase-free glass or polypropylene centrifuge tube (not supplied). add the appropriate volume of Buffer RLT (see Table 6) to the cell-culture dish. Vortex or pipet to mix. Note: Incomplete loosening of the cell pellet may lead to inefficient lysis and reduced RNA yields. Both effects may reduce RNA yield.1–0. 2. and proceed to step 2. and ensure that no cell clumps are visible before proceeding to step 3. and centrifuge at 300 x g for 5 min. affecting the conditions for binding of RNA to the RNeasy membrane. and proceed to step 3. Collect the lysate with a rubber policeman. Table 6. Table 5. Aspirate the medium. After the cells detach from the dish or flask.25% trypsin in PBS. Aspirate the PBS. Completely aspirate the supernatant. To trypsinize and collect cells: Determine the number of cells.
Reuse the collection tube in step 6. and centrifuge for 15 s at _8000 x g (_10. Homogenize the lysate according to step 3a. This does not affect the procedure. Close the lid gently. Note: The volume of lysate may be less than 350 µl or 600 µl due to loss during homogenization. and centrifuge for 15 s at _8000 x g (_10. Add 700 µl Buffer RW1 to the RNeasy spin column. Do not centrifuge. Discard the flow-through. Discard the flow-through after each centrifugation. 37 .Transfer up to 700 µl of the sample. centrifuge successive aliquots in the same RNeasy spin column. Note: When purifying RNA from certain cell lines. 6. Be sure to empty the collection tube completely.000 rpm).Dish (cm) <6 6–10 Animal Cells Spin diameter Volume of Buffer RLT (µl_ 350 600 3. 4. If the sample volume exceeds 700 µl.000 rpm) to wash the spin column membrane. Discard the flow-through. or 3c. Homogenize the lysate for 30 s using a rotor–stator homogenizer. including any precipitate that may have formed.9 mm diameter) fitted to an RNase-free syringe. 3a. Proceed to step 4. carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. 3b. Spin Note: After centrifugation. 3c. to an RNeasy spin column placed in a 2 ml collection tube. and centrifuge for 2 min at full speed. Reuse the collection tube in step 7. 3b. Proceed to step 4. Pass the lysate at least 5 times through a blunt 20-gauge needle (0. precipitates may be visible after addition of ethanol. Add 1 volume of 70% ethanol to the homogenized lysate. Pipet the lysate directly into a QIAshredder spin column placed in a 2 ml collection tube. Close the lid gently. and mix well by pipetting. 5. Proceed to step 4.
and discard the old collection tube with the flow-through. Close the lid gently.1. or if residual flow-through remains on the outside of the RNeasy spin column after step 8.000 rpm) to elute the RNA. the RNA yield will be 15–30% less than that obtained using a second volume of RNase-free water. Otherwise. 3.3 Isolation of total RNA from Sera Samples: QIAamp Viral RNA Mini Spin Protocol: 38 . Residual ethanol may interfere with downstream reactions. or using the eluate from step 10 (if high RNA concentration is required). If using the eluate from step 10. 8. and centrifuge for 1 min at _8000 x g(_10. repeat step 10 using another 30–50 µl RNasefree water. Close the lid gently. carryover of ethanol will occur. Optional: Place the RNeasy spin column in a new 2 ml collection tube. Add 30–50 µl RNase-free water directly to the spin column membrane. The long centrifugation dries the spin column membrane. and centrifuge for 15 s at _8000 x g (_10. Close the lid gently. but the final RNA concentration will be higher.4. Reuse the collection tube from step 10. Add 500 µl Buffer RPE to the RNeasy spin column.000 rpm) to wash the spin column membrane. Reuse the collection tube in step 8. 9. and centrifuge for 2 min at _8000 x g (_10. If the expected RNA yield is >30 µg. 11. 10.5 ml collection tube (supplied). carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Ensure that ethanol is added to Buffer RPE before use. Close the lid gently. Perform this step to eliminate any possible carryover of Buffer RPE. Place the RNeasy spin column in a new 1. Discard the flow-through. ensuring that no ethanol is carried over during RNA elution. Add 500 µl Buffer RPE to the RNeasy spin column.7.000 rpm) to wash the spin column membrane. and centrifuge at full speed for 1 min. Note: After centrifugation. Note: Buffer RPE is supplied as a concentrate.
1. and discard the tube containing the filtrate. repeat this step until all of the lysate has been loaded onto the spin column. Briefly centrifuge the falcon tube to remove drops from the inside of the lid. Place the QIAamp spin column into a clean 2-ml collection tube. Carefully open the QIAamp spin column. Centrifuge at full speed for 1 min.5-ml microcentrifuge tube (not provided). . or to eliminate any chance of possible Buffer AW2 carryover. Place the QIAamp spin column in a clean 1. and mix by pulse-vortexing for 15 sec. 3. Discard the old collection tube containing the filtrate. 6. Centrifuge at 6000 x g (8000 rpm) for 1 min. 14. perform step 9a. Close the cap. If the sample volume was greater than 140 μl. and discard the tube containing the filtrate. and then continue with step 10. Close the cap and centrifuge at full speed (20. 39 .Place the QIAamp spin column in a new 2-ml collection tube and discard the old collection tube with the filtrate. Mix by pulse-vortexing for 15 sec. Continue directly with step 10. 5. and centrifuge at 6000 x g (8000 rpm) for 1 min. Close the cap. and add 500 μl of Buffer AW2. Carefully open the QIAamp spin column and add 30 μl of Buffer AVE equilibrated to room temperature. Carefully open the QIAamp spin column. 9. Add 1120 μl of ethanol (96–100%) to the sample. briefly centrifuge the 1. 2. Close the cap. 7. Pipet 1120 μl of prepared Buffer AVL ( viral lysis buffer) and add 112 μl of Carrier RNA already dissolved in AVE Buffer in a sterilized Falcon. After mixing. and add 500 μl of Buffer AW1(washing buffer). Place the QIAamp spin column in a clean 2-ml collection tube (provided). 4. 10.000 rpm) for 3 min. Add 280μl serum to the Buffer AVL/Carrier RNA in the falcon tube. and repeat step 6. 8.5-ml microcentrifuge tube to remove drops from inside the lid. and incubate at room temperature for 1 min.000 x g. Carefully open the QIAamp spin column. and centrifuge at 6000 x g (8000 rpm) for 1 min. Incubate at room temperature (15–25°C) for 10 min. Viral RNA is stable for up to one year when stored at –20°C or –70°C. Carefully apply 630 μl of the solution from step 5 to the QIAamp spin column (in a 2-ml collection tube) without wetting the rim.
4.24 µg/µl = 40 X 0. The gel was casted in a fume hood.043= 1.0) 40 mM sodium acetate 5 MM EDTA (PH 8.052= 2. 40 .3.72 µg/µl 3. N1 3.031= 1.2 RNA Quantification • • Purified RNA was diluted to 1:1000 using DEPC water. 5 x formaldehyde gel-running buffer was added to give final concentrations of 1X and 15% formaldehyde.3 Denaturing Gel Electrophoresis of RNA 5x formaldehyde gel-running buffer: 0.1M MOPS (PH 7. Quantification of RNA concentration was done by reading the absorbance at nm/280 nm. C3 = 40 X 0.0) The gel was prepared by melting the appropriate amount of agarose in water and cooled to 60°C. and allowed to set for at least 30 minutes at RT. TE 13 2.4.08 µg/µl = 40 X 0. • Concentration of RNA was determined byConcentration of RNA = 40 X A260 /volume of RNA (µl ) assayed 260 nm and the contamination of the RNA sample was determined by the absorbance ratio at 260 Total RNA concentration of some of the samples: 1.
The gel was electrophoresed in 1x formaldehyde gel-running buffer at 3-4 V/cm.25% bromophenol blue 0.5µ l .5 µg/ml in 0.25% xylene cyanol FF Before loading the samples.1 M ammonium acetate) for 30-45 minutes.0) 0.5µl 2.0µl Samples were heat denatured at 65°C for 15 minutes to remove secondary structures. Formaldehyde gel-loading buffer: 50% glycerol 1mM EDTA (pH 8.5µl 10. then immediately put on ice 41 DEPC water 3.Samples were prepared as follows: RNA (upto 10 µg) 5 x formaldehyde gel-running buffer Formaldehyde Formamide 4. the gel was prerun for 5 minutes at 5 V/cm. 3.0µl 3. At the end of the run.4 Preparation of cDNA from reverse transcription reaction At 70oC for 5 min.4. chilled immediately on ice and loaded on the gel by mixing with 2µl of sterile. DEPC-treated formaldehyde gel-loading buffer immediately after heat denaturation. the gel was stained with ethidium bromide (0.
5 Polymerase Chain Reaction 42 .2 µ g/µ l) Total RNA (10-16 µg) 5X Reaction buffer dNTPs (10 mM) RT enzyme 1. Primers used in the study Human β -actin (product size: 646 bp) 5'-CAG CCA TGT ACG TTG CTA TCC AG -3' 5'-GTT TCG TGG ATG CCA CAG GAC-3' Annealing Temperature.4.56oC 3.5µ l 8µ l 4µ l 2µ l 1µ l At 250 C for 5 min.62 oC BMI-1 (product size: 799 bp) 5’ CAG CAA TGA CTG TGA TGC ACT 3’ 5’ GCC CAA TGC TTA TGT CCA CT 3’ Annealing Temperature. 70oC for 10 min. 42oC for 1 hr. 25o C for 5 min.Random Hexamers (0.
0 µ l 2.6 Agarose Gel Electrophoresis 43 .5 µ l 0.2 µ l 3. (from RT) Forward Primer Reverse Primer Taq Polymerase (5 U /µ l) Total Volume 12. ∞ 35 Cycles 3. 10 min. 45 sec.4.0 µ l 0.2 µ l 20 µ l General Parameters 940C 940C Tm 720C 720C 40C 5 min.5 µ l 0.0 µ l 0. 1 min.6 µ l 1. 60 sec.dH20 10x PCR buffer dNTP (25 µ M) MgCl2 cDNA Prep.
1 RNA ISOLATION RNA was isolated from the cancerous cell line TE 13. 1). 2 µl of low range Fermentas 100 bp DNA ladder was also loaded.Agraose gels (1%) prepared in 1X Tris Borate EDTA buffer (TBE) with ethidium bromide (EtBr) solution added to give a final concentration of 5µ g/ml were casted. 28s and 18s were obtained on the gel (Fig. DNA samples were prepared by addition of 5X loading dye and loaded into the wells of the gel. ESCC tissues as well as matched distant normal esophageal tissues and the serum samples of patients with carcinoma using the TRI Reagent method as described earlier. 44 .RESULTS 4. 4. Once the gel had been set and the gel plate support was placed into the running apparatus filled with 1X running buffer to just cover the wells. The quality of RNA was checked on 1% agarose gel. The gels were run at a constant voltage of 80V for 1-2 hrs and the integrity of PCR products was viewed using a UV transilluminator. Two intact bands viz.
and TE13 cell line using the cDNA prepared for each of the isolated RNA. cancerous (C) esophageal tissues and TE 13 cell line.2 REVERSE TRANSCRIPTION-PCR ANALYSIS OF HOUSE KEEPING GENE β -Actin The expression of the house keeping gene i. 4. The integrity of the β -Actin PCR product (646 bp in size as per the primer used) indicated the successful synthesis of cDNA. 45 . β -Actin was studied in different Esophageal tissues (both cancerous and the matched normal).28 s 18 s N1 C1 N27 C27 N7 C7 N30 TE13 Figure 1: Representative 1% agarose gel showing presence of intact RNA from normal (N). (Fig.e. 2).
TE13: positive control. C: ESCC patient cancer tissues. NS: Normal sera 46 . N: ESCC patient adjacent normal tissues.646b p Ladder N27 C27 N28 C28 C32 N32 C31 TE13 Figure 2: Representative 1% agarose gel showing amplification of β -Actin from the same RT reaction as used for the genes study: 4.e. β -Actin. adjacent matched normal tissues and TE13 cell line by semi-quantitative RT-PCR using the same RT reaction as used for the study of housekeeping gene i. Ladder TE13 C7 C32 N32 C28 N28 S9 NS Figure 3: Representative 1% agarose gel showing expression of Bmi-1 gene Ladder: 100 bp ladder. S: Sera of ESCC patients.3 REVERSE TRANSCRIPTION-PCR ANALYSIS OF STEM CELL RELATED GENE IN ESCCs Expression of the stem cell gene. Bmi-1 was studied in esophageal squamous cell carcinomas. The same RT reaction enables the comparison of the expression of stem cell genes with the expression of β -Actin gene in normal and cancer tissues.
A.A N.A. Bmi1 expressio S.A N.A.A.TABLE 1: Clinicopathological parameters of Esophageal Squamous Cell Carcinoma patient and the expression of cancer stem cell related gene analyzed.A. + + + + + + + N.NO. N.A N.A. AGE GENDER alcohol smoker 1 52 M yes yes 1 28 M yes yes 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 64 M 55 F 52 M 46 M 57 M YES YES NO NO YES YES NO YES NO path status MDSCC WDSCC WDSCC SUPERFICIAL FRAGMENTS SUPERFICIAL FRAGMENTS ADINOCARCINOMA NO REPORT NO REPORT CARCINOMA STRATIFIED n in tissue sera N. N.A NA + + - + + + + + + + + N. - NO NO YES MDSCC DYSPLASTIC FRAGMENTS NO REPORT + - 47 . N. + N. 65 M 72 M 28 F 34 M 55 F 65 F 80 M 64 M 45 M 54 F 55 M 50 M 56 M 60 F 45 M 54 M SQUAMOUS ADINOCARCINOMA MDSCC MDSCC + + + - YES NO NO NO NO NO NO REPORT MDSCC PDSCC SCC HYPERPLASIA MDSCC _ N.A.
BMI-1 mRNA expression was observed in 17/23 (70. the level of expression was not very high though as compared to normal sera collected from a healthy individual. Densitometric analysis would be further required to see the difference in the levels of expression in cancerous and adjacent non cancerous tissues. the expression of BMI-1 was analyzed in esophageal cancer cell culture (TE13)... Thus. The over expression of the gene was correlated with the clinicopathological parameters of the samples viz. 2002. also taken as positive control for RT-PCR analysis.. 2006). and it is significant to identify a biological genetic molecular marker related to its pathophysiological processes. 2006. This may be due to the fact that the endoscopically collected normal tissues are from a site adjacent to the tumor and may be subjected to field cancerization. As there is heterogeneity in the origin of cells present in surgically resected ESCC tissue specimens. 2009). rather than the DNA sequence. gender. Interestingly. Sparmann et al.. 5.Discussion ESCC is a major cause of morbidity and mortality worldwide (Fisichella et al. Bmi1. age. are not true normals. the heritable changes in gene expression that occur in chromatin structure including DNA methylation. BMI-1 mRNA expression was found to be elevated in 5 ESCC sera out of 11 samples.8%) cases. histone post-translational modifications and nucleosomal remodeling.. Epigenetic aberrations. are involved in cancer development(Fraga et al. It showed expression in all the adjacent non cancerous matched tissues. all the hyperplastic and dysplastic tissues analyzed for the expression showed expression of BMI1 transcripts. is a 48 . Quina et al. the first PcG protein found. histopathology and habits of the ESCC patients.
2008).. and may be of diagnostic value. Bmi1 mRNA expression was significantly higher in the ESCC samples. 2006. Shilatifard et al. 2007). Scheijen et al. higher expression was observed in samples with higher grade carcinoma which is in accordance with the study by Kozakowski et al. men are found to be at a higher risk then women (Enzinger et al. are not true normals... However. The level of Bmi1 mRNA expression was found to be elevated in all the dysplastic and hyperplastic tissue samples analyzed along with true carcinomas.. The Bmi1 mRNA expression was also found to be elevated in some sera samples of ESCC patients as compared to normal sera... 1999. also high levels of expression were seen in the adjacent non-cancerous tissues. its expression needs to be validated in a larger cohort for better understanding of clinical significance of its expression in ESCCs. Thus.. whether the expression of Bmi1 in sera can be used in non-invasive diagnosis of ESCC. 49 . and this could be due to the low presence of circulating Bmi1 mRNA in ESCC patients or due to the low levels of total circulating mRNAs in sera. needs further validation in larger cohort using quantitative PCR techniques. This indicated that Bmi1 plays an important role in the development of ESCC. Sparmann et al. Thus. The level of expression of Bmi1 mRNA can be correlated to the histology of the samples. In this study. Bmi1 expression is also found inversely correlated with the differentiation grade of clear cell carcinoma and is involved in tumor progression (Kozakowski et al. 2003). the expression of BMI1 gene in ESCC and its impact on ESCC patients was investigated. 2008.chromatin modifier implicated in the tumorigenesis through negatively regulating the gene expression such as the INK4A locus. The risk for cancer also increases with age and most of the patients are above 50 years of age. which is thought to regulate p53 and the Rb signaling pathway in cooperation with c-myc (Jacobs et al. the percentage of sera samples with elevated expression was less than 50%. 2006).. 1999. Bmi1-deficient tumors may be less aggressive because they have fewer stem cells (Dirks et al. this may be rendered to the fact that the endoscopically collected normal tissues are from a site adjacent to the tumor and may be subjected to field cancerization.
A Benito-Hernandez. MS Wicha. (2003). hence this study can be used as a reference for an extensive study on Bmi1 mRNA in sera. are needed to confirm the conclusions. Some previous studies have shown that Bmi1 expression is a potential escape mechanism and associated with markedly increased likelihood of treatment failure and disease relapse after surgery (Liu et al. Advances in the treatment of esophageal carcinoma. Proc Natl Acad Sci USA 100:3983–3988. 2. Thus.. 278294. 50 . References 1. SJ Morrison and MF Clarke. Till date no study has been done on using circulating mRNAs of Bmi1 as markers for ESCC. Whether Bmi1 can be used for accurate prediction of ESCC and its potential chemosensitivity to current pharmaceutical treatment needs further study. Qin et al have found that down-regulation of Bmi-1 enhances 5-fluorouracil-induced apoptosis in nasopharyngeal carcinoma cells and have suggested that the combination of 5-FU treatment and Bmi-1 depletion might be a potential clinical strategy for cancer chemotherapy.. further investigations on larger series of ESCC patients. prospective identification of tumorigenic breast cancer cells. 2006). including clinical follow-up and novel molecular techniques.The mechanisms of Bmi1 up-expression that induce adverse pathological and clinical features in ESCC patients are poorly understood. Gastroenterologist. 5: 1997.Berezovska et al. 6. Al-Hajj M. 2005.
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PBS Buffer 54g 27. 2. TBE Buffer 5x (1 litre) Tris Base Boric Acid EDTA (0.2g 1.0 3.24g in 800 ml of distilled H2O. pH 8.5 g 20ml 1x PBS NaCl KCl Na2HPO4 KH2PO4 8g 0.2g 2. Add H2O to 1 liter.4 with HCl. Abbreviation ADC Adenocarcinoma 56 . Adjust the pH to 7.0G Added 10% FBS and 1X Ciprofloxacin to the media.5 M).44g 0.RPMI Media NaHCO3 pH 7.2 16. Sterilize by autoclave.
ASR DEPC EC EDTA ESCC EtBr MDSCC mg ml MMLV nm PCR PDSCC RT Taq TBE TNM ug WDSCC Annual Survival Rate Diethyl Pyro Carbonate Esophageal Squamous Ethylene Diamine Tetra Acetic acid Esophageal Squamous Cell Carcinoma Ethidium Bromide Moderately differentiated Squamous Cell Carcinoma milli gram milli litre Moloney Murine Leukemia Virus nano metre Polymerase Chain Reaction Poorly differentiated Squamous Cell Carcinoma Reverse Transription Thermus acquaticus Tris Borate EDTA buffer Tumor. Metastasis micro gram Well differentiated Squamous Cell Carcinoma 57 . Node.