This chapter focuses on the nucleic acid blotting techniques for virus detection. The Southern an... more This chapter focuses on the nucleic acid blotting techniques for virus detection. The Southern and Northern blotting procedures have evolved with some general alterations and numerous case-specific ones. The chapter discusses the techniques to prepare and process nucleic acids from cells and tissues for use in filter hybridization assays, along with a variety of techniques to separate nucleic acid pools to hybridization and the preparation of hybridization probes, and methods for detection and quantification of hybridization signals. Specific examples of these technologies show the detection of nucleic acids from human immunodeficiency virus type 1. The ability to identify specific nucleic acid sequences within the genome or as part of the pool of transcribed RNAs has proven to be an invaluable tool to all molecular biologists. Southern, Northern, and dot/slot blot procedures are currently widely used for detection of clinically relevant viral nucleic acids. The blotting-based procedures are highly sensitive, reproducible, and relatively inexpensive. In addition, Southern blotting of products of the polymerase chain reaction (PCR) is an important adjunct procedure used to increase the sensitivity of PCR and to confirm the identity of the amplified DNA.
Archives of Otolaryngology Head Neck Surgery, Apr 1, 2009
Objective: To assess whether chronic periodontitis history predicts human papillomavirus (HPV) st... more Objective: To assess whether chronic periodontitis history predicts human papillomavirus (HPV) status in patients with base of tongue cancers.
Human papillomavirus 16 (HPV-16) infection and tobacco use are associated with human oropharyngea... more Human papillomavirus 16 (HPV-16) infection and tobacco use are associated with human oropharyngeal cancers. We conducted a study of the role of HPV and tobacco use in base of the tongue (BOT) cancers. DNA from 34 such cancers was subjected to HPV-16 and HPV-18-specific polymerase chain reaction analysis. Demographic and clinicopathologic data were obtained from each patient's medical record. HPV-16 was detected in 68% of tumors. Tobacco use was the only factor found to be significantly associated with HPV status. Tumors from 100% of patients who had never used tobacco tested positive for HPV, compared with only 56% of those who had ever used tobacco (Fisher exact test, p = 0.024). All tumors were associated with either tobacco use or HPV infection. These findings are consistent with the hypothesis that either tobacco use or HPV infection is necessary to the etiology of BOT tumors, and they suggest that tongue base carcinoma may be prevented by combining HPV vaccination with tobacco avoidance.
Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics, Feb 1, 2005
Genomic instability is a fundamental characteristic of solid tumors, and understanding genomic in... more Genomic instability is a fundamental characteristic of solid tumors, and understanding genomic instability should significantly clarify the process of tumorigenesis. We adapted the sampling technique of inter-(simple sequence repeat) PCR [inter-(SSR) PCR] to measure genetic alterations between simple sequence repeats in colorectal tumors. It becomes important to precisely define both normal and altered inter-(SSR) PCR products. BLAT searches of 131 cloned inter-(SSR) PCR sequences reveal that inter-(SSR) PCR products are located on almost all the chromosomes except chromosome Y, indicating that inter-(SSR) PCR samples a representative diverse range of the genome. We confirm that a change in the pattern of the inter-(SSR) PCR products as seen on gel electrophoresis reflects a true alteration within the genome.
Objectives: Lactic acid producing streptococci and lactobacilli are part of the commensal flora a... more Objectives: Lactic acid producing streptococci and lactobacilli are part of the commensal flora and have been shown to exert protective effects against chronic inflammatory diseases and cancer. These beneficial bacteria increase mucosal protection, bind/degrade carcinogens, produce anti-tumorigenic compounds, and promote cell-mediated immune response. Dental caries is the demineralization of tooth structures by lactic acid formed by bacterial fermentation of dietary sugars. Studies suggest that cariogenic bacteria Streptococcus mutans and Lactobacillus casei, stimulate a highly polarized cell-mediated immunity. Methods: We performed a case-control study to test the association between dental caries and head and neck squamous cell carcinoma (HNSCC). Cases were patients newly diagnosed with primary HNSCC between 1999 and 2005 at Roswell Park Cancer Institute. Controls were new patients seen during the same time period as the cases but not diagnosed with cancer. Edentulous patients and...
Objectives: Human papillomavirus (HPV) has emerged as an independent risk factor for a subset of ... more Objectives: Human papillomavirus (HPV) has emerged as an independent risk factor for a subset of head and neck squamous cell carcinoma (HNSCC). HPV has a specific tropism for epithelial basal cells, usually gains access through ulcerations, and its replication is closely associated with epithelial proliferation. Periodontitis is a significant source of bacteria and inflammatory cytokines in saliva. Mucosal injury and proliferative state caused by chronic infection/inflammation may facilitate HPVs acquisition and persistence in the oral mucosa. The aim of this study was to test whether periodontitis predicts tumor HPV status. Methods: We performed a case-control study. The study population consisted of patients newly diagnosed with primary HNSCC between 1999 and 2005 at Roswell Park Cancer Institute, Buffalo, NY. All patients who had both tumor samples and dental records available were included into the study. Periodontitis history was measured by alveolar bone loss (ABL) from panora...
Objective: Human papillomavirus 16 (HPV-16) has been accepted as an independent risk factor for a... more Objective: Human papillomavirus 16 (HPV-16) has been accepted as an independent risk factor for a subset of head and neck squamous cell carcinoma (HNSCC). Paradoxically, studies suggest improved survival of patients with HPV-positive tumors. The objective of this study was to assess the association between tumor HPV status and stage in HNSCC patients. Methods: We performed a cross-sectional study in a comprehensive cancer center in Upstate, NY. The study population consisted of all newly diagnosed primary HNSCC patients between 1999 and 2007 for whom the information on tumor stage and HPV status was available. Patients with a history of cancer were excluded. HPV-16 DNA was identified by polymerase chain reaction from paraffin embedded tumor samples. Odds ratios (OR) and their 95% confidence intervals (CI) were estimated from multiple logistic regression analysis. Results: A total of 112 incident HNSCC cases were included. From those, 23 (20.5%) were diagnosed with early (I/II), and ...
Objectives: Incidence of HPV-positive head and neck cancers (HNSCC) has increased unpredictably i... more Objectives: Incidence of HPV-positive head and neck cancers (HNSCC) has increased unpredictably in parallel to declining smoking rates since early 70s. The objective of this study was to assess the effects of smoking and tumor HPV status on HNSCC phenotype. Methods: We performed a retrospective analysis of existing data in a comprehensive cancer center. The study population consisted of newly diagnosed primary HNSCC patients between 1999 and 2007 for whom the information on HPV and smoking status was available. Edentulous, immunocompromised, and those with a history of cancer were excluded. HPV-16 DNA was identified by polymerase chain reaction from paraffin embedded tumor samples. Results: A total of 124 HNSCC cases were included. Prevalence of HPV-positive tumors was 50/124 (40.3%), and the prevalence of smoking was 114/124 (91.9%). Compared to never smokers, smokers were in average 4 years older at diagnosis (59 vs. 55 years), were 2 times more likely to have a late stage (TNM II...
Objective: Dental caries and periodontitis are two plaque-related but distinct diseases in terms ... more Objective: Dental caries and periodontitis are two plaque-related but distinct diseases in terms of etiology and pathogenesis. Periodontitis, associated with gram-negative anaerobic bacteria, is characterized by Th2- and Th17-polarized immune responses, which are associated with increased risk of cancer. Dental caries, associated with gram-positive lactic acid bacteria, elicit potent Th1 immune response promoting CD8+ T-cell response,which is associated with decreased risk of cancer. The purpose of the present study was to test the association of oral squamous cell carcinoma (OSCC) with dental caries and periodontitis. Method: We performed a case-control study. Cases were patients newly diagnosed with primary OSCC between 1999 and 2007 at Roswell Park Cancer Institute. Controls were new patients seen during the same time as cases but not diagnosed with cancer. Patients with immunodeficiency or previous history of cancer were excluded. Dental caries, periodontitis and missing teeth w...
Objective: Dental caries and periodontitis are two plaque-related but distinct diseases in terms ... more Objective: Dental caries and periodontitis are two plaque-related but distinct diseases in terms of etiology and pathogenesis. Periodontitis, associated with gram-negative anaerobic bacteria, is characterized by Th2- and Th17-polarized immune responses, which are associated with increased risk of cancer. Dental caries, associated with gram-positive lactic acid bacteria, elicit potent Th1 immune response promoting CD8+ T-cell response,which is associated with decreased risk of cancer. The purpose of the present study was to test the association of oral squamous cell carcinoma (OSCC) with dental caries and periodontitis. Method: We performed a case-control study. Cases were patients newly diagnosed with primary OSCC between 1999 and 2007 at Roswell Park Cancer Institute. Controls were new patients seen during the same time as cases but not diagnosed with cancer. Patients with immunodeficiency or previous history of cancer were excluded. Dental caries, periodontitis and missing teeth w...
Familial cancer syndromes have revealed important fundamental features regarding how all cancers ... more Familial cancer syndromes have revealed important fundamental features regarding how all cancers arise through destabilization of the genome, such that somatic evolution can select for the disruption of critical cellular coordinating and regulatory features. The authors examine those cellular genes and systems whose normal role is to preserve genomic integrity and relate them to the genetic foundations of heritable cancers. By examining how these cellular systems normally function, how family cancer genes are able to affect the process of tumor progression can be learned. In so doing, a clearer picture of how sporadic cancers arise is additionally gained.
Familial cancer syndromes have revealed important fundamental features regarding how all cancers ... more Familial cancer syndromes have revealed important fundamental features regarding how all cancers arise through destabilization of the genome, such that somatic evolution can select for the disruption of critical cellular coordinating and regulatory features. The authors examine those cellular genes and systems whose normal role is to preserve genomic integrity and relate them to the genetic foundations of heritable cancers. By examining how these cellular systems normally function, how family cancer genes are able to affect the process of tumor progression can be learned. In so doing, a clearer picture of how sporadic cancers arise is additionally gained.
Objectives. The object of this study is to measure genomic instability in papillary thyroid cance... more Objectives. The object of this study is to measure genomic instability in papillary thyroid cancer and correlate these measurements with known clinical prognosticators such as patient age, tumor size, histologic subtype, and three commonly used thyroid risk assessment indices. A secondary objective of this study was to use the measurements of genomic instability to estimate the number of mutational events present in the papillary thyroid cancer genome.
Genomic instability is a fundamental characteristic of solid tumors, and understanding genomic in... more Genomic instability is a fundamental characteristic of solid tumors, and understanding genomic instability should significantly clarify the process of tumorigenesis. We adapted the sampling technique of inter-(simple sequence repeat) PCR [inter-(SSR) PCR] to measure genetic alterations between simple sequence repeats in colorectal tumors. It becomes important to precisely define both normal and altered inter-(SSR) PCR products. BLAT searches of 131 cloned inter-(SSR) PCR sequences reveal that inter-(SSR) PCR products are located on almost all the chromosomes except chromosome Y, indicating that inter-(SSR) PCR samples a representative diverse range of the genome. We confirm that a change in the pattern of the inter-(SSR) PCR products as seen on gel electrophoresis reflects a true alteration within the genome.
We have used genome-wide allelotyping with 348 polymorphic autosomal markers spaced, on average, ... more We have used genome-wide allelotyping with 348 polymorphic autosomal markers spaced, on average, 10 cM apart to quantitate the extent of intrachromosomal instability in 59 human sporadic colorectal carcinomas. We have compared instability measured by this method with that measured by inter-(simple sequence repeat) PCR and microsatellite instability assays. Instability quantitated by fractional allelic loss rates was found to be independent of that detected by microsatellite instability analyses but was weakly associated with that measured by inter-(simple sequence repeat) PCR. A set of seven loci were identified that were most strongly associated with elevated rates of fractional allelic loss and/or inter-(simple sequence repeat) PCR instability; these seven loci were on chromosomes 3, 8, 11, 13, 14, 18, and 20. A lesser association was seen with two loci flanking p53 on chromosome 17. Coordinate loss patterns for these loci suggest that at least two separate sets of cooperating loc...
Colorectal carcinogenesis is a multistep process with an apparently orderly progression from beni... more Colorectal carcinogenesis is a multistep process with an apparently orderly progression from benign tissue to invasive malignancy and metastases. Yet at the genome level, a considerably more chaotic situation exists, with order arising through the process of natural selection in the midst of genomic instability. Major pathways for colorectal carcinogenesis begin with suppressor loss or acquisition of a mutator phenotype, but there are other pathways known and yet to be described. These pathways result in the natural selection of cells with unstable genomes leading to malignancy and metastases.
VL30 elements are associated with cancer by their overexpression in rodent malignancies, their in... more VL30 elements are associated with cancer by their overexpression in rodent malignancies, their induction in a fibroblast response to anoxia which shares features with the malignant phenotype, and their presence recombined into Harvey murine sarcoma virus (HaSV) and Kirsten murine sarcoma virus. These sarcoma viruses contain ras oncogenes flanked on both sides by retrotransposon VL30 element sequences, in turn flanked by mouse leukemia virus sequences. Three very basic questions have existed about the VL30 element sequences found in sarcoma viruses: (i) how did they become recombined, (ii) what are their exact boundaries, and (iii) why are they there? To help decipher the nature of VL30 elements in sarcoma viruses, we examined VL30 clones isolated from an anoxic fibroblast cDNA library and independently by polymerase chain reaction cloning from rat cell DNA. Sequence comparisons with HaSV revealed that HaSV was formed by the substitution of 0.7 kb of VL30 sequences by 0.9 kb of c-Ha-...
Fischer rat embryo fibroblasts subjected to temporary anoxia followed by an aerobic recovery peri... more Fischer rat embryo fibroblasts subjected to temporary anoxia followed by an aerobic recovery period show genomic instability in the form of highly elevated CAD gene amplification rates. As revealed by flow cytometric analysis this is associated with DNA breakage in vivo, followed by repair during the recovery period. Such genomic instability parallels expression of a M(r) 29,000/31,000 endonuclease; this enzyme requires no added divalent metal ion and has a pH optimum of about 6.5. The same endonuclease was found to be expressed within healing wounds and in four of ten human colorectal cancers but was not seen in eight normal colorectal tissue samples. Our results indicate that DNA breakage resulting from endogenous endonuclease activity can have a substantial effect in modulating genomic instability.
Cells exposed to hypoxia undergo substantial changes in gene expression generally associated with... more Cells exposed to hypoxia undergo substantial changes in gene expression generally associated with metabolic adaptation and increasing oxygen delivery. In contrast, responses distinct from those elicited by hypoxia are induced in anoxic fibroblasts; this includes activation of a set of VL30 elements. The responses seen in anoxically cultured fibroblasts are expressed physiologically in vivo during the anaerobic phase of wound healing. A fundamental question is whether transcriptional regulatory pathways utilized during anoxia are distinct from those already characterized for hypoxic cells. We report here the isolation of a 14-bp sequence within a VL30 retrotransposon promoter which mediates its anoxia responsiveness. Analyses of the protein complexes binding to this sequence demonstrated the presence of two distinct inducible DNA binding activities. The first is present in both hypoxic and anoxic fibroblasts and is indistinguishable from hypoxia-inducible factor 1. The second activit...
ABSTRACT A murine leukemia virus was cloned by a method which yielded cell clones each chronicall... more ABSTRACT A murine leukemia virus was cloned by a method which yielded cell clones each chronically infected with a virus clone. One of these produced virus which had an abnormal protein pattern characterized by increased amounts of certain proteins normally present in trace amounts. Immunoreplica analysis showed that these proteins were related to the major capsid protein p30 and suggested that they were uncleaved precursor proteins or other high-molecular-weight proteins derived from the gag gene. The major species of RNA in this clone were the same size as those in a clone producing normal virus. When uninfected cells were infected with virus produced by this clone, the progeny had a normal protein pattern. Finally, this clone was abnormal in its growth characteristics in that the cells died rapidly upon becoming confluent. This cell clone, therefore, seems to have a cell-determined deficiency in the processing of leukemia virus proteins. Electron microscopy showed that this cell line produced an abnormally high proportion of virus particles which had an immature morphology.
This chapter focuses on the nucleic acid blotting techniques for virus detection. The Southern an... more This chapter focuses on the nucleic acid blotting techniques for virus detection. The Southern and Northern blotting procedures have evolved with some general alterations and numerous case-specific ones. The chapter discusses the techniques to prepare and process nucleic acids from cells and tissues for use in filter hybridization assays, along with a variety of techniques to separate nucleic acid pools to hybridization and the preparation of hybridization probes, and methods for detection and quantification of hybridization signals. Specific examples of these technologies show the detection of nucleic acids from human immunodeficiency virus type 1. The ability to identify specific nucleic acid sequences within the genome or as part of the pool of transcribed RNAs has proven to be an invaluable tool to all molecular biologists. Southern, Northern, and dot/slot blot procedures are currently widely used for detection of clinically relevant viral nucleic acids. The blotting-based procedures are highly sensitive, reproducible, and relatively inexpensive. In addition, Southern blotting of products of the polymerase chain reaction (PCR) is an important adjunct procedure used to increase the sensitivity of PCR and to confirm the identity of the amplified DNA.
Archives of Otolaryngology Head Neck Surgery, Apr 1, 2009
Objective: To assess whether chronic periodontitis history predicts human papillomavirus (HPV) st... more Objective: To assess whether chronic periodontitis history predicts human papillomavirus (HPV) status in patients with base of tongue cancers.
Human papillomavirus 16 (HPV-16) infection and tobacco use are associated with human oropharyngea... more Human papillomavirus 16 (HPV-16) infection and tobacco use are associated with human oropharyngeal cancers. We conducted a study of the role of HPV and tobacco use in base of the tongue (BOT) cancers. DNA from 34 such cancers was subjected to HPV-16 and HPV-18-specific polymerase chain reaction analysis. Demographic and clinicopathologic data were obtained from each patient's medical record. HPV-16 was detected in 68% of tumors. Tobacco use was the only factor found to be significantly associated with HPV status. Tumors from 100% of patients who had never used tobacco tested positive for HPV, compared with only 56% of those who had ever used tobacco (Fisher exact test, p = 0.024). All tumors were associated with either tobacco use or HPV infection. These findings are consistent with the hypothesis that either tobacco use or HPV infection is necessary to the etiology of BOT tumors, and they suggest that tongue base carcinoma may be prevented by combining HPV vaccination with tobacco avoidance.
Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics, Feb 1, 2005
Genomic instability is a fundamental characteristic of solid tumors, and understanding genomic in... more Genomic instability is a fundamental characteristic of solid tumors, and understanding genomic instability should significantly clarify the process of tumorigenesis. We adapted the sampling technique of inter-(simple sequence repeat) PCR [inter-(SSR) PCR] to measure genetic alterations between simple sequence repeats in colorectal tumors. It becomes important to precisely define both normal and altered inter-(SSR) PCR products. BLAT searches of 131 cloned inter-(SSR) PCR sequences reveal that inter-(SSR) PCR products are located on almost all the chromosomes except chromosome Y, indicating that inter-(SSR) PCR samples a representative diverse range of the genome. We confirm that a change in the pattern of the inter-(SSR) PCR products as seen on gel electrophoresis reflects a true alteration within the genome.
Objectives: Lactic acid producing streptococci and lactobacilli are part of the commensal flora a... more Objectives: Lactic acid producing streptococci and lactobacilli are part of the commensal flora and have been shown to exert protective effects against chronic inflammatory diseases and cancer. These beneficial bacteria increase mucosal protection, bind/degrade carcinogens, produce anti-tumorigenic compounds, and promote cell-mediated immune response. Dental caries is the demineralization of tooth structures by lactic acid formed by bacterial fermentation of dietary sugars. Studies suggest that cariogenic bacteria Streptococcus mutans and Lactobacillus casei, stimulate a highly polarized cell-mediated immunity. Methods: We performed a case-control study to test the association between dental caries and head and neck squamous cell carcinoma (HNSCC). Cases were patients newly diagnosed with primary HNSCC between 1999 and 2005 at Roswell Park Cancer Institute. Controls were new patients seen during the same time period as the cases but not diagnosed with cancer. Edentulous patients and...
Objectives: Human papillomavirus (HPV) has emerged as an independent risk factor for a subset of ... more Objectives: Human papillomavirus (HPV) has emerged as an independent risk factor for a subset of head and neck squamous cell carcinoma (HNSCC). HPV has a specific tropism for epithelial basal cells, usually gains access through ulcerations, and its replication is closely associated with epithelial proliferation. Periodontitis is a significant source of bacteria and inflammatory cytokines in saliva. Mucosal injury and proliferative state caused by chronic infection/inflammation may facilitate HPVs acquisition and persistence in the oral mucosa. The aim of this study was to test whether periodontitis predicts tumor HPV status. Methods: We performed a case-control study. The study population consisted of patients newly diagnosed with primary HNSCC between 1999 and 2005 at Roswell Park Cancer Institute, Buffalo, NY. All patients who had both tumor samples and dental records available were included into the study. Periodontitis history was measured by alveolar bone loss (ABL) from panora...
Objective: Human papillomavirus 16 (HPV-16) has been accepted as an independent risk factor for a... more Objective: Human papillomavirus 16 (HPV-16) has been accepted as an independent risk factor for a subset of head and neck squamous cell carcinoma (HNSCC). Paradoxically, studies suggest improved survival of patients with HPV-positive tumors. The objective of this study was to assess the association between tumor HPV status and stage in HNSCC patients. Methods: We performed a cross-sectional study in a comprehensive cancer center in Upstate, NY. The study population consisted of all newly diagnosed primary HNSCC patients between 1999 and 2007 for whom the information on tumor stage and HPV status was available. Patients with a history of cancer were excluded. HPV-16 DNA was identified by polymerase chain reaction from paraffin embedded tumor samples. Odds ratios (OR) and their 95% confidence intervals (CI) were estimated from multiple logistic regression analysis. Results: A total of 112 incident HNSCC cases were included. From those, 23 (20.5%) were diagnosed with early (I/II), and ...
Objectives: Incidence of HPV-positive head and neck cancers (HNSCC) has increased unpredictably i... more Objectives: Incidence of HPV-positive head and neck cancers (HNSCC) has increased unpredictably in parallel to declining smoking rates since early 70s. The objective of this study was to assess the effects of smoking and tumor HPV status on HNSCC phenotype. Methods: We performed a retrospective analysis of existing data in a comprehensive cancer center. The study population consisted of newly diagnosed primary HNSCC patients between 1999 and 2007 for whom the information on HPV and smoking status was available. Edentulous, immunocompromised, and those with a history of cancer were excluded. HPV-16 DNA was identified by polymerase chain reaction from paraffin embedded tumor samples. Results: A total of 124 HNSCC cases were included. Prevalence of HPV-positive tumors was 50/124 (40.3%), and the prevalence of smoking was 114/124 (91.9%). Compared to never smokers, smokers were in average 4 years older at diagnosis (59 vs. 55 years), were 2 times more likely to have a late stage (TNM II...
Objective: Dental caries and periodontitis are two plaque-related but distinct diseases in terms ... more Objective: Dental caries and periodontitis are two plaque-related but distinct diseases in terms of etiology and pathogenesis. Periodontitis, associated with gram-negative anaerobic bacteria, is characterized by Th2- and Th17-polarized immune responses, which are associated with increased risk of cancer. Dental caries, associated with gram-positive lactic acid bacteria, elicit potent Th1 immune response promoting CD8+ T-cell response,which is associated with decreased risk of cancer. The purpose of the present study was to test the association of oral squamous cell carcinoma (OSCC) with dental caries and periodontitis. Method: We performed a case-control study. Cases were patients newly diagnosed with primary OSCC between 1999 and 2007 at Roswell Park Cancer Institute. Controls were new patients seen during the same time as cases but not diagnosed with cancer. Patients with immunodeficiency or previous history of cancer were excluded. Dental caries, periodontitis and missing teeth w...
Objective: Dental caries and periodontitis are two plaque-related but distinct diseases in terms ... more Objective: Dental caries and periodontitis are two plaque-related but distinct diseases in terms of etiology and pathogenesis. Periodontitis, associated with gram-negative anaerobic bacteria, is characterized by Th2- and Th17-polarized immune responses, which are associated with increased risk of cancer. Dental caries, associated with gram-positive lactic acid bacteria, elicit potent Th1 immune response promoting CD8+ T-cell response,which is associated with decreased risk of cancer. The purpose of the present study was to test the association of oral squamous cell carcinoma (OSCC) with dental caries and periodontitis. Method: We performed a case-control study. Cases were patients newly diagnosed with primary OSCC between 1999 and 2007 at Roswell Park Cancer Institute. Controls were new patients seen during the same time as cases but not diagnosed with cancer. Patients with immunodeficiency or previous history of cancer were excluded. Dental caries, periodontitis and missing teeth w...
Familial cancer syndromes have revealed important fundamental features regarding how all cancers ... more Familial cancer syndromes have revealed important fundamental features regarding how all cancers arise through destabilization of the genome, such that somatic evolution can select for the disruption of critical cellular coordinating and regulatory features. The authors examine those cellular genes and systems whose normal role is to preserve genomic integrity and relate them to the genetic foundations of heritable cancers. By examining how these cellular systems normally function, how family cancer genes are able to affect the process of tumor progression can be learned. In so doing, a clearer picture of how sporadic cancers arise is additionally gained.
Familial cancer syndromes have revealed important fundamental features regarding how all cancers ... more Familial cancer syndromes have revealed important fundamental features regarding how all cancers arise through destabilization of the genome, such that somatic evolution can select for the disruption of critical cellular coordinating and regulatory features. The authors examine those cellular genes and systems whose normal role is to preserve genomic integrity and relate them to the genetic foundations of heritable cancers. By examining how these cellular systems normally function, how family cancer genes are able to affect the process of tumor progression can be learned. In so doing, a clearer picture of how sporadic cancers arise is additionally gained.
Objectives. The object of this study is to measure genomic instability in papillary thyroid cance... more Objectives. The object of this study is to measure genomic instability in papillary thyroid cancer and correlate these measurements with known clinical prognosticators such as patient age, tumor size, histologic subtype, and three commonly used thyroid risk assessment indices. A secondary objective of this study was to use the measurements of genomic instability to estimate the number of mutational events present in the papillary thyroid cancer genome.
Genomic instability is a fundamental characteristic of solid tumors, and understanding genomic in... more Genomic instability is a fundamental characteristic of solid tumors, and understanding genomic instability should significantly clarify the process of tumorigenesis. We adapted the sampling technique of inter-(simple sequence repeat) PCR [inter-(SSR) PCR] to measure genetic alterations between simple sequence repeats in colorectal tumors. It becomes important to precisely define both normal and altered inter-(SSR) PCR products. BLAT searches of 131 cloned inter-(SSR) PCR sequences reveal that inter-(SSR) PCR products are located on almost all the chromosomes except chromosome Y, indicating that inter-(SSR) PCR samples a representative diverse range of the genome. We confirm that a change in the pattern of the inter-(SSR) PCR products as seen on gel electrophoresis reflects a true alteration within the genome.
We have used genome-wide allelotyping with 348 polymorphic autosomal markers spaced, on average, ... more We have used genome-wide allelotyping with 348 polymorphic autosomal markers spaced, on average, 10 cM apart to quantitate the extent of intrachromosomal instability in 59 human sporadic colorectal carcinomas. We have compared instability measured by this method with that measured by inter-(simple sequence repeat) PCR and microsatellite instability assays. Instability quantitated by fractional allelic loss rates was found to be independent of that detected by microsatellite instability analyses but was weakly associated with that measured by inter-(simple sequence repeat) PCR. A set of seven loci were identified that were most strongly associated with elevated rates of fractional allelic loss and/or inter-(simple sequence repeat) PCR instability; these seven loci were on chromosomes 3, 8, 11, 13, 14, 18, and 20. A lesser association was seen with two loci flanking p53 on chromosome 17. Coordinate loss patterns for these loci suggest that at least two separate sets of cooperating loc...
Colorectal carcinogenesis is a multistep process with an apparently orderly progression from beni... more Colorectal carcinogenesis is a multistep process with an apparently orderly progression from benign tissue to invasive malignancy and metastases. Yet at the genome level, a considerably more chaotic situation exists, with order arising through the process of natural selection in the midst of genomic instability. Major pathways for colorectal carcinogenesis begin with suppressor loss or acquisition of a mutator phenotype, but there are other pathways known and yet to be described. These pathways result in the natural selection of cells with unstable genomes leading to malignancy and metastases.
VL30 elements are associated with cancer by their overexpression in rodent malignancies, their in... more VL30 elements are associated with cancer by their overexpression in rodent malignancies, their induction in a fibroblast response to anoxia which shares features with the malignant phenotype, and their presence recombined into Harvey murine sarcoma virus (HaSV) and Kirsten murine sarcoma virus. These sarcoma viruses contain ras oncogenes flanked on both sides by retrotransposon VL30 element sequences, in turn flanked by mouse leukemia virus sequences. Three very basic questions have existed about the VL30 element sequences found in sarcoma viruses: (i) how did they become recombined, (ii) what are their exact boundaries, and (iii) why are they there? To help decipher the nature of VL30 elements in sarcoma viruses, we examined VL30 clones isolated from an anoxic fibroblast cDNA library and independently by polymerase chain reaction cloning from rat cell DNA. Sequence comparisons with HaSV revealed that HaSV was formed by the substitution of 0.7 kb of VL30 sequences by 0.9 kb of c-Ha-...
Fischer rat embryo fibroblasts subjected to temporary anoxia followed by an aerobic recovery peri... more Fischer rat embryo fibroblasts subjected to temporary anoxia followed by an aerobic recovery period show genomic instability in the form of highly elevated CAD gene amplification rates. As revealed by flow cytometric analysis this is associated with DNA breakage in vivo, followed by repair during the recovery period. Such genomic instability parallels expression of a M(r) 29,000/31,000 endonuclease; this enzyme requires no added divalent metal ion and has a pH optimum of about 6.5. The same endonuclease was found to be expressed within healing wounds and in four of ten human colorectal cancers but was not seen in eight normal colorectal tissue samples. Our results indicate that DNA breakage resulting from endogenous endonuclease activity can have a substantial effect in modulating genomic instability.
Cells exposed to hypoxia undergo substantial changes in gene expression generally associated with... more Cells exposed to hypoxia undergo substantial changes in gene expression generally associated with metabolic adaptation and increasing oxygen delivery. In contrast, responses distinct from those elicited by hypoxia are induced in anoxic fibroblasts; this includes activation of a set of VL30 elements. The responses seen in anoxically cultured fibroblasts are expressed physiologically in vivo during the anaerobic phase of wound healing. A fundamental question is whether transcriptional regulatory pathways utilized during anoxia are distinct from those already characterized for hypoxic cells. We report here the isolation of a 14-bp sequence within a VL30 retrotransposon promoter which mediates its anoxia responsiveness. Analyses of the protein complexes binding to this sequence demonstrated the presence of two distinct inducible DNA binding activities. The first is present in both hypoxic and anoxic fibroblasts and is indistinguishable from hypoxia-inducible factor 1. The second activit...
ABSTRACT A murine leukemia virus was cloned by a method which yielded cell clones each chronicall... more ABSTRACT A murine leukemia virus was cloned by a method which yielded cell clones each chronically infected with a virus clone. One of these produced virus which had an abnormal protein pattern characterized by increased amounts of certain proteins normally present in trace amounts. Immunoreplica analysis showed that these proteins were related to the major capsid protein p30 and suggested that they were uncleaved precursor proteins or other high-molecular-weight proteins derived from the gag gene. The major species of RNA in this clone were the same size as those in a clone producing normal virus. When uninfected cells were infected with virus produced by this clone, the progeny had a normal protein pattern. Finally, this clone was abnormal in its growth characteristics in that the cells died rapidly upon becoming confluent. This cell clone, therefore, seems to have a cell-determined deficiency in the processing of leukemia virus proteins. Electron microscopy showed that this cell line produced an abnormally high proportion of virus particles which had an immature morphology.
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Papers by Daniel Stoler