Cancer stem cell model in oral squamous cell carcinoma

The concept of "field cancerization" describes the presence of histological abnormal tissue surrounding oral squamous cell carcinoma (OSCC). Molecular model of multistep carcinogenesis indicates that an accumulation of genetic alterations forms the basis for the OSCC progression with genetic heterogeneity. Furthermore, we reviewed cancer stem cell (CSC) model, which suggests functional heterogeneity in the tumor mass and current supporting evidence in OSCC. According to CSC model, prevention from carcinogen exposure and eliminating the particular CSCs instead of targeting tumor mass could help obtain a more long-lasting therapeutic effect.     

Genomic instability in silica- and cadmium chloride-transformed BALB/c-3T3 and tumor cell lines by random amplified polymorphic DNA analysis

Our earlier studies using random amplified polymorphic DNA (RAPD) analysis have shown genetic instability in human lung cancer tissues. Here we have investigated the potential for genetic instability in silica- and cadmium chloride (CdCl2)-transformed BALB/c-3T3 cell lines. Non-transformed, transformed BALB/c-3T3 cells, and tumor cell lines (obtained by injecting nude mice with transformed cell lines) were analyzed for genomic changes. DNAs from 10 different transformed clones and their corresponding tumor cell lines were amplified individually by RAPD analysis using 10 arbitrary primers. DNA from non-transformed BALB/c-3T3 cells was used as a control to compare genetic alterations, if any, between non-transformed, transformed and tumor cell populations. PCR products from RAPD were electrophoretically separated on agarose gels and the banding profiles were visualized by ethidium bromide staining. Five of the 10 primers tested revealed genomic changes in silica-transformed cell lines when compared to non-transformed BALB/c-3T3 cells. Comparison of all 10 transformed and tumor cell lines showed varied degrees of genomic changes using all 10 primers. CdCl2-transformed cell lines displayed fewer genomic changes, only three of 10 primers showed a positive result. CdCl2-transformed cells and their corresponding tumor cell lines showed specific banding pattern differences in six of the 10 samples tested with six of the 10 primers. Changes in band intensity were the most commonly observed changes both in silica- and CdCl2-transformed and tumor cell lines. The results seem to indicate a progressive change in genomic rearrangements which may directly or indirectly be associated with progression of tumorigenesis.     

Fields and field cancerization: the preneoplastic origins of cancer: asymptomatic hyperplastic fields are precursors of neoplasia, and their progression to tumors can be tracked by saturation density in culture

Most basic research on cancer concerns genetic changes in benign and malignant tumors. Yet evidence indicates that the majority of the mutations in tumors occur in the preneoplastic field stage of their development. That early stage is represented by grossly invisible, broad regions of "field cancerization" which have not, heretofore, been operationally analyzed in cell culture. Conditions are described for quantitating preneoplasia by increased saturation density followed by progression to transformation. These parameters are driven by Darwinian selection of spontaneously occurring, cumulative mutations, in accordance with recent genomic analyses of human cancer, just as it is in the evolution of species. The cell culture model will allow correlation of the preneoplastic increases in saturation density with genetic changes, and development of methods for demarcating fields during surgery so that they can be excised along with the tumor, thereby reducing the possibility of recurrence at the site.     

Raman exfoliative cytology for prognosis prediction in oral cancers: A proof of concept study

Oral cancer is associated with high rates of recurrence, attributable to field cancerization. Early detection of advanced field changes that can potentially progress to carcinoma can facilitate timely intervention and can lead to improved prognosis. Previous in vivo studies have successfully detected advanced field effects in oral cancers. Raman exfoliative cytology has previously shown to differentiate normal, oral pre‐cancer and cancers. The present study explores Raman‐exfoliative‐cytology‐based detection of field effects. Exfoliated cells were collected from tumor (n = 16) and contralateral‐normal appearing mucosa (n = 16) of oral cancer patients, and healthy tobacco habitués (n = 20). After spectral acquisition, specimens were Pap‐stained for cytological evaluation. Data analysis, by Principal Component Analysis and Principal Component‐Linear Discriminant Analysis, indicate several spectral‐misclassifications between contralateral normal and tumor, which were investigated and correlated with spectral, cytological and clinical outcomes. A qualitative analysis by grouping patients with number of misclassifications with tumor (Group 1: 0, Group 2: 1 and Group 3: >1) was explored. Group 3 with highest misclassifications showed spectral and cytological similarity to tumor group — one patient was a case of early inoperable residual disease, despite clear margins on histopathology. Thus, these misclassifications could be indicative of cancer field changes, and can prospectively help to identify patients susceptible to recurrences .     

Mice expressing SV40 T antigen directed by the intestinal trefoil factor promoter develop tumors resembling human small cell carcinoma of the colon

The colonic epithelium contains three major types of mature cells, namely, absorptive, goblet, and enteroendocrine cells. These cells are maintained by a complex process of cell renewal involving progenitor and stem cells, and colon cancers develop when this process goes awry. Much is known about the genetic and epigenetic changes that occur in cancer; however, little is known as to the specific cell types involved in carcinogenesis. In this study, we expressed the SV40 Tag oncogene in the intestinal epithelium under the control of an intestinal trefoil factor (ITF) promoter. This caused tumor formation in the proximal colon with remarkable efficiency. ITFTag tumors were rapidly growing, multifocal, and invasive. ITFTag tumor cells express synaptophysin and contain dense core secretory granules, markers of neuroendocrine differentiation. The cell type involved in the early steps of ITFTag tumorigenesis was studied by examining partially transformed crypts that contained populations of both normal and dysplastic cells. The dysplastic cell population always expressed both Tag and synaptophysin. Cells expressing Tag alone were never observed; however, normal enteroendocrine cells expressing synaptophysin but not Tag were readily visualized. This suggests that ITFTag tumor cells originate from the enteroendocrine cell lineage following a transforming event that results in Tag expression. ITFTag tumors closely resemble human small cell carcinomas of the colon, suggesting the possibility that these tumors might be derived from the enteroendocrine cell lineage as well.     

Deciphering the molecular mechanism of the cancer formation by chromosome structural dynamics

Cancer reflects the dysregulation of the underlying gene network, which is strongly related to the 3D genome organization. Numerous efforts have been spent on experimental characterizations of the structural alterations in cancer genomes. However, there is still a lack of genomic structural-level understanding of the temporal dynamics for cancer initiation and progression. Here, we use a landscape-switching model to investigate the chromosome structural transition during the cancerization and reversion processes. We find that the chromosome undergoes a non-monotonic structural shape-changing pathway with initial expansion followed by compaction during both of these processes. Furthermore, our analysis reveals that the chromosome with a more expanding structure than those at both the normal and cancer cell during cancerization exhibits a sparse contact pattern, which shows significant structural similarity to the one at the embryonic stem cell in many aspects, including the trend of contact probability declining with the genomic distance, the global structural shape geometry and the spatial distribution of loci on the chromosome. In light of the intimate structure-function relationship at the chromosomal level, we further describe the cell state transition processes by the chromosome structural changes, suggesting an elevated cell stemness during the formation of the cancer cells. We show that cell cancerization and reversion are highly irreversible processes in terms of the chromosome structural transition pathways, spatial repositioning of chromosomal loci and hysteresis loop of contact evolution analysis. Our model draws a molecular-scale picture of cell cancerization from the chromosome structural perspective. The process contains initial reprogramming towards the stem cell followed by the differentiation towards the cancer cell, accompanied by an initial increase and subsequent decrease of the cell stemness.     

Mapping yeast origins of replication via single-stranded DNA detection

Studies in th Saccharomyces cerevisiae have provided a framework for understanding how eukaryotic cells replicate their chromosomal DNA to ensure faithful transmission of genetic information to their daughter cells. In particular, S. cerevisiae is the first eukaryote to have its origins of replication mapped on a genomic scale, by three independent groups using three different microarray-based approaches. Here we describe a new technique of origin mapping via detection of single-stranded DNA in yeast. This method not only identified the majority of previously discovered origins, but also detected new ones. We have also shown that this technique can identify origins in Schizosaccharomyces pombe, illustrating the utility of this method for origin mapping in other eukaryotes.     

Genome-wide profiling of gene amplification and deletion in cancer

Accumulations of genetic changes in somatic cells induce phenotypic transformations leading to cancer. Among these genetic changes, gene amplification and deletion are most frequently observed in several kinds of cancers. Amplification of oncogene and/or deletion of tumor suppressor gene, together with dysfunction of the gene by point mutation, are the main causes of cancer. Genome-wide analysis of amplification and deletion of genes in cancers is basic to resolving the mechanisms of carcinogenesis. Comparative genomic hybridization (CGH) developed in 1992 has been utilized to identify DNA copy number abnormalities in various kind of cancers and several reports have shown its usefulness in screening of the genes involved in carcinogenesis, and also in the identification of prognostic factors in cancer. We have shown that 1q23 gain is associated with neuroblastomas that are resistant to aggressive treatment, and have poor prognosis, and 1q and 13q gains are possibly related to drug resistance in ovarian cancers. Recently, the "rough draft" of the human genome was reported and we are ready to utilize the vast information on genomic sequences in cancer research. Moreover, microarray technology enables us to analyze more than ten thousand genes at a time and revealed genetic abnormalities in cancers at a genome-wide level. By combination of microarray and CGH, a powerful screening method for oncogenes and tumor suppressor genes in cancers, called array-CGH, has been developed by several groups. In this article, we overview these genome-wide analytical methods, CGH and array-CGH, and discuss their potential in molecular characterization of cancers.     

A new theory of the origin of cancer: quantum coherent entanglement, centrioles, mitosis, and differentiation

Malignant cells are characterized by abnormal segregation of chromosomes during mitosis ("aneuploidy"), generally considered a result of malignancy originating in genetic mutations. However, recent evidence supports a century-old concept that maldistribution of chromosomes (and resultant genomic instability) due to abnormalities in mitosis itself is the primary cause of malignancy rather than a mere byproduct. In normal mitosis chromosomes replicate into sister chromatids which are then precisely separated and transported into mirror-like sets by structural protein assemblies called mitotic spindles and centrioles, both composed of microtubules. The elegant yet poorly understood ballet-like movements and geometric organization occurring in mitosis have suggested guidance by some type of organizing field, however neither electromagnetic nor chemical gradient fields have been demonstrated or shown to be sufficient. It is proposed here that normal mirror-like mitosis is organized by quantum coherence and quantum entanglement among microtubule-based centrioles and mitotic spindles which ensure precise, complementary duplication of daughter cell genomes and recognition of daughter cell boundaries. Evidence and theory supporting organized quantum states in cytoplasm/nucleoplasm (and quantum optical properties of centrioles in particular) at physiological temperature are presented. Impairment of quantum coherence and/or entanglement among microtubule-based mitotic spindles and centrioles can result in abnormal distribution of chromosomes, abnormal differentiation and uncontrolled growth, and account for all aspects of malignancy. New approaches to cancer therapy and stem cell production are suggested via non-thermal laser-mediated effects aimed at quantum optical states of centrioles.     

The unc-86 gene product couples cell lineage and cell identity in C. elegans

The C. elegans gene unc-86 is required in several distinct neuroblast lineages for daughter cells to become different from their mothers, and is also required for the specification of particular neural identities. Consistent with the fact that unc-86 encodes a POU domain protein, we find that the unc-86 protein is localized to the nucleus. In the affected lineages, unc-86 protein appears within a few minutes after cell division in the nuclei of those daughter cells that are transformed by unc-86 mutations. Thus, expression of unc-86 protein is dependent on cell lineage. unc-86 protein is not asymmetrically segregated at further divisions. unc-86 protein also appears shortly after cell division in the nuclei of particular identified differentiating neurons; at least some of these neurons are nonfunctional in unc-86 mutants.     

The life history of 21 breast cancers

Cancer evolves dynamically as clonal expansions supersede one another driven by shifting selective pressures, mutational processes, and disrupted cancer genes. These processes mark the genome, such that a cancer's life history is encrypted in the somatic mutations present. We developed algorithms to decipher this narrative and applied them to 21 breast cancers. Mutational processes evolve across a cancer's lifespan, with many emerging late but contributing extensive genetic variation. Subclonal diversification is prominent, and most mutations are found in just a fraction of tumor cells. Every tumor has a dominant subclonal lineage, representing more than 50% of tumor cells. Minimal expansion of these subclones occurs until many hundreds to thousands of mutations have accumulated, implying the existence of long-lived, quiescent cell lineages capable of substantial proliferation upon acquisition of enabling genomic changes. Expansion of the dominant subclone to an appreciable mass may therefore represent the final rate-limiting step in a breast cancer's development, triggering diagnosis.     

Live imaging of tumor initiation in zebrafish larvae reveals a trophic role for leukocyte-derived PGE₂

Epidemiology studies and clinical trials have suggested that the use of non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, can significantly reduce the incidence of and mortality associated with many cancers, and upregulation of the COX2-PGE(2) pathway in tumor microenvironments might drive several aspects of cancer progression. For these reasons, the mechanisms linking COX blockade and cancer prevention have long been an area of active investigation. During carcinogenesis, COX-2 is expressed both by malignant epithelial cells and by tumor-associated stromal cells, including macrophages, but the observation that NSAIDs are most effective in cancer prevention in APC(min/+) mice if the mice are treated from conception suggests that the COX-2/PGE(2) pathway might also be critical at the earliest stages of tumor development. In this study we take advantage of the translucency and genetic tractability of zebrafish larvae to investigate the involvement of inflammatory cells at cancer initiation, when transformed cells first arise in tissues. We previously showed that innate immune cells supply early transformed cells with proliferative cues and, by using complementary pharmacological and genetic experiments, we now show that prostaglandin E(2) (PGE(2)) is the trophic signal required for this expansion of transformed cells. Our in vivo observations at these early stages of cancer initiation provide a potential mechanistic explanation for why long-term use of low doses of NSAIDs, including aspirin, might reduce cancer onset.     

Fates of the blastomeres of the 32-cell-stage Xenopus embryo

A detailed fate map of all of the progeny derived from each of the blastomeres of the 32-cell-stage South African clawed frog embryo (Xenopus laevis), which were selected for stereotypic cleavages, is presented. Individual blastomeres were injected with horseradish peroxidase and all of their descendants in the late tailbud embryo (stages 32 to 34) were identified after histochemical processing of serial tissue sections and whole-mount preparations. The progeny of each blastomere were distributed characteristically, both in phenotype and location. Most organs were populated largely by the descendants of particular sets of blastomeres, the progeny of each often being restricted to defined spatial addresses. Thus, the descendants of any one blastomere were distinct and predictable when embryos were preselected for stereotypic cleavages. However, variations among embryos were common and the frequencies with which one may expect organs to contain progeny from any particular blastomere are reported. The differences in the fates of the 16-cell-stage blastomeres and their 32-cell-stage daughter blastomeres are outlined and can be grouped into three general categories. The two daughter cells may give rise to equal numbers of cells in a particular organ, one daughter cell may give rise to many more of the cells in an organ derived from the mother blastomere, or one daughter cell may give rise to all of the progeny in an organ derived from the mother blastomere. Thus, cell fates are segregated during cleavage stages in both symmetric and asymmetric manners, and the lineages exhibit a diversification mode (G. S. Stent, 1985, Philos. Trans R. Soc. London Ser. B 312, 3-19) of cell division.     

The contribution of endogenous sources of DNA damage to the multiple mutations in cancer

There is increasing evidence that most human cancers contain multiple mutations. By the time a tumor is clinically detectable it may have accumulated tens of thousands of mutations. In normal cells, mutations are rare events occurring at a rate of 10(-10) mutations per nucleotide per cell per generation. We have argued that the mutation rates exhibited by normal human cells are insufficient to account for the large number of mutations found in human cancers, and therefore, that an early event in tumorigenesis is the development of a mutator phenotype. In normal cells, spontaneous and induced DNA damage is balanced by multiple pathways for DNA repair, and most DNA damage is repaired without error. However, in tumor cells this balance may be shifted such that damage overwhelms the repair capacity, resulting in the accumulation of multiple mutations. Our hypothesis is that multiple random mutations occur during carcinogenesis. The sequential mutations that are observed in some human tumors result from selective events required for tumor progression. We consider the possibility that endogenous sources of DNA damage, in particular oxidative DNA damage, may contribute to genomic instability and to a mutator phenotype in some tumors. Endogenous and environmental sources of reactive oxygen species (ROS) are abundant. In tumor cells, antioxidant or DNA repair capacity may be insufficient to compensate for the production of ROS, and these endogenous ROS may be capable of damaging DNA and inducing mutations in critical DNA stability genes. The possibility that oxidative DNA damage could be a significant source of the genomic instability characteristic of human cancers is exciting, because it may be feasible to modulate the extent of oxidative damage through antioxidant therapy. The use of antioxidants to reduce the extent of molecular damage by ROS could delay the progression of cancer.     

Characterization of murine cell lines from Diethylstilbestrol-Induced uterine endometrial Adenocarcinomas

Neonatal treatment with estrogens is associated with development of uterine adenocarcinomas in CD-1 mice. Treatment with the synthetic estrogen diethylstilbestrol (DES) on Days 1 to 5 after birth results in 90% incidence of these hormonedependent lesions in 18-mo.-old mice. Three cell lines were established from these DES-associated tumors. Each of these cell lines exhibited morphologic and ultrastructural characteristics of transformed epithelial cells, including an increased nuclear:ytoplasmic ratio, enlarged and irregular nuclei with multiple nucleoli and areas of chromatin condensation, positive staining for cytokeratin, desmosomes, and microvilli. After subcutaneous injection into nude mice, all three cell lines formed solid tumors within 4 wk. Although the primary uterine tumors and tumor transplants in nude mice had been shown to be estrogen-dependent and estrogen-receptor positive, neither the monolayer growth nor the tumorigenicity of any of the three cell lines in this study was enhanced by or dependent on estrogen. Estrogen receptor levels were low in early and intermediate passage cells. Allele-specific oligonucleotide hybridization analysis of PCR-amplified cell line DNA revealed no point mutations in the 12th, 13th, or 61st codons of the K-ras or H-ras protooncogenes. Southern analysis revealed no changes in genomic organization of the putative tumor suppressor gene DCC, but demonstrated a three-to four-fold amplification of the c-myc gene in one cell line. Expression of c-myc RNA was concomitantly increased in the same cell line. These three transformed cell lines represent the end point in the process of hormone-associated tumorigenesis and as such should prove useful in investigating the molecular changes and the mechanisms involved in hormonal carcinogenesis.     

Human cell transformation in the study of sunlight-induced cancers in the skin of man

Human cell transformation provides a powerful approach to understanding--at the cellular and molecular levels--induction of cancers in the skin of man. A principal approach to this problem is the direct transformation of human skin cells by exposure to ultraviolet and/or near-UV radiation. The frequency of human cells transformed to anchorage independence increases with radiation exposure; the relative transforming efficiencies of different wavelengths implies that direct absorption by nucleic acids is a primary initial event. Partial reversal of potential transforming lesions by photoreactivation suggests that pyrimidine dimers, as well as other lesions, are important in UV transformation of human cells. Human cells can also be transformed by transfection with cloned oncogenes, or with DNAs from tumors or tumor cell lines. Cells treated by the transfection procedure (but without DNA) or cells transfected with DNAs from normal mammalian cells or tissues show only background levels of transformation. Human cells can be transformed to anchorage-independent growth by DNAs ineffective in transformation of NIH 3T3 cells (including most human skin cancers), permitting the analysis of oncogenic molecular changes even in tumor DNAs difficult or impossible to analyze in rodent cell systems.     

3p21, 5q21, 9p21 and 17p13 allelic deletions accumulate in the dysplastic spectrum of laryngeal carcinogenesis and precede malignant transformation

A tissue field of somatic genetic alterations precede the histopathological phenotypic changes of carcinoma. Loss of Heterozygosity (LOH) at the sites of known or putative tumor suppressor genes is a common genetic abnormality detected in precancerous conditions. These genomic changes could be of potential use in the diagnosis and prognosis of pre-malignant laryngeal lesions. Recently the concept of laryngeal intraepithelial neoplasia (LIN) was introduced. To evaluate patients with an increased risk of developing invasive laryngeal carcinoma via a dysplasia-carcinoma progression we investigated 102 microdissected cell populations. Cell populations were procured from 15 laryngectomy specimens with different peritumoral histological changes adjacent to the squamous cell carcinoma cells and 15 laryngeal endoscopic biopsies with no evidence of malignant transformation in a 6-10-year follow-up period. Histological diagnoses were subdivided into keratosis without dysplasia (KWD), with mild dysplasia (LIN 1), with moderate dysplasia (LIN 2), and with severe dysplasia or carcinoma in situ (LIN 3). Microsatellite analysis was performed with the aim of studying LOH of 5q21 (APC), 9p21 (p16), 3p21 and 17p13 (p53) chromosomal regions. Frequent allelic losses were found in carcinoma cells at p53 (54%), p16 (66%), 3p21(87%) and 5q21(58%). Identical LOH patterns were determined in 100% of the LIN3 peritumoral cells, 60% of LIN2, 50% of LIN 1 and 25% of KWD. In contrast, histologically normal mucosae, KWD and LIN1 lesions without malignant progression showed no allelic loss. These results show that dysplasia correlates with LOH at 3p21, 5q21, 9p21 and 17p13 in early laryngeal carcinogenesis. These genomic changes in pre-malignant laryngeal lesions could be of potential use as markers for cancer risk assessment.     

Early Embryonic Chromosome Instability Results in Stable Mosaic Pattern in Human Tissues

The discovery of copy number variations (CNV) in the human genome opened new perspectives on the study of the genetic causes of inherited disorders and the aetiology of common diseases. Here, a single-cell-level investigation of CNV in different human tissues led us to uncover the phenomenon of mitotically derived genomic mosaicism, which is stable in different cell types of one individual. The CNV mosaic ratios were different between the 10 individuals studied. However, they were stable in the T lymphocytes, immortalized B lymphoblastoid cells, and skin fibroblasts analyzed in each individual. Because these cell types have a common origin in the connective tissues, we suggest that mitotic changes in CNV regions may happen early during embryonic development and occur only once, after which the stable mosaic ratio is maintained throughout the differentiated tissues. This concept is further supported by a unique study of immortalized B lymphoblastoid cell lines obtained with 20 year difference from two subjects. We provide the first evidence of somatic mosaicism for CNV, with stable variation ratios in different cell types of one individual leading to the hypothesis of early embryonic chromosome instability resulting in stable mosaic pattern in human tissues. This concept has the potential to open new perspectives in personalized genetic diagnostics and can explain genetic phenomena like diminished penetrance in autosomal dominant diseases. We propose that further genomic studies should focus on the single-cell level, to better understand the aetiology of aging and diseases mediated by somatic mutations.