On the potential origin and characteristics of cancer stem cells.

The 'cancer stem cell' hypothesis has pointed to a specific target for new cancer therapies. The hypothesis is based on the observation that only the 'cancer stem cell' among the other heterogeneous cancer cells can sustain the growth of the cancer. The goal is to identify biomarkers of 'cancer stem cells' to distinguish them from the 'cancer non-stem cells' and normal adult tissue-specific stem cells. This analyst posits a hypothesis that, although all cancers originated from a single cell, there exist two types of 'cancer stem cells' either by the 'Stem Cell hypothesis' or from the 'De-differentiation hypothesis'. It is proposed that there exist two different 'cancer stem cells'. Some 'cancer stem cells' (a) lack the expression of connexins or gap junction genes and lack any form of gap junctional intercellular communication (GJIC) or (b) they have the expressed connexin-coded proteins for functional GJIC but are dysfunctional by some expressed oncogene. This is consistent with the Loewenstein hypothesis that a universal characteristic of cancer cells is they do not have growth control, nor terminally differentiate. This review speculates the normal organ-specific adult stem cell, that is 'initiated', is the origin of the 'cancer stem cells' with expressed Oct4A gene and no expressed connexin genes; whereas the other cancer stem cell has no expressed Oct4A genes but expressed connexin gene, whose coded protein is dysfunctional. Hence. both types of 'cancer stem cells' lack GJIC, for two different reasons, the selective therapies have to be different for these different cell types.

Categorizing the characteristics of human carcinogens: a need for specificity.

The International Agency for Research on Cancer (IARC) has recently proposed employing "ten key characteristics of human carcinogens" (TKCs) to determine the potential of agents for harmful effects. The TKCs seem likely to confuse the unsatisfactory correlation from testing regimes that have ignored the differences evident when cellular changes are compared in short and long-lived species, with their very different stem cell and somatic cell phylogenies. The proposed characteristics are so broad that their use will lead to an increase in the current unacceptably high rate of false positives. It could be an informative experiment to take well-established approved therapeutics with well-known human safety profiles and test them against this new TKC paradigm. Cancers are initiated and driven by heritable and transient changes in gene expression, expand clonally, and progress via additional associated acquired mutations and epigenetic alterations that provide cells with an evolutionary advantage. The genotoxicity testing protocols currently employed and required by regulation, emphasize testing for the mutational potential of the test agent. Two-year, chronic rodent cancer bioassays are intended to test for the entire spectrum of carcinogenic transformation. The use of cytotoxic doses causing increased, sustained cell proliferation that facilitates accumulated genetic damage leads to a high false-positive rate of tumor induction. Current cancer hazard assessment protocols and weight-of-the-evidence analysis of agent-specific cancer risk align poorly with the pathogenesis of human carcinoma and so need modernization and improvement in ways suggested here.

Applicability of Scrape Loading-Dye Transfer Assay for Non-Genotoxic Carcinogen Testing.

Dysregulation of gap junction intercellular communication (GJIC) is recognized as one of the key hallmarks for identifying non-genotoxic carcinogens (NGTxC). Currently, there is a demand for in vitro assays addressing the gap junction hallmark, which would have the potential to eventually become an integral part of an integrated approach to the testing and assessment (IATA) of NGTxC. The scrape loading-dye transfer (SL-DT) technique is a simple assay for the functional evaluation of GJIC in various in vitro cultured mammalian cells and represents an interesting candidate assay. Out of the various techniques for evaluating GJIC, the SL-DT assay has been used frequently to assess the effects of various chemicals on GJIC in toxicological and tumor promotion research. In this review, we systematically searched the existing literature to gather papers assessing GJIC using the SL-DT assay in a rat liver epithelial cell line, WB-F344, after treating with chemicals, especially environmental and food toxicants, drugs, reproductive-, cardio- and neuro-toxicants and chemical tumor promoters. We discuss findings derived from the SL-DT assay with the known knowledge about the tumor-promoting activity and carcinogenicity of the assessed chemicals to evaluate the predictive capacity of the SL-DT assay in terms of its sensitivity, specificity and accuracy for identifying carcinogens. These data represent important information with respect to the applicability of the SL-DT assay for the testing of NGTxC within the IATA framework.

Ready to go 3D? A semi-automated protocol for microwell spheroid arrays to increase scalability and throughput of 3D cell culture testing.

3-dimensional (3D) cell cultures are being increasingly recognized as physiologically more relevant in vitro models than traditional monolayer cultures, because they better mimic in vivo-like microenvironment, cell-cell and cell-extracellular matrix interactions. Nevertheless, the broader use of 3D models might be limited by requirements for special consumables, equipment, or skills for 3D cell cultures, and by their limited throughput and scalability. In this study, we optimized and adapted a commercially available agarose-micromolding technique to produce scaffold-free spheroid cultures. Brightfield microscopy was used for routine nondestructive and noninvasive evaluation of spheroid formation and growth. The workflow is compatible with manual, as well as high speed automated microscopic image acquisition, and it is supplemented with an in-house developed macro 'Spheroid_Finder' for open source software Fiji to facilitate rapid automated image analysis. This protocol was used to characterize and quantify spheroid formation and growth of two different hepatic cell lines, hTERT immortalized, but non-cancerous, adult human liver stem cell line HL1-hT1, and human hepatocellular carcinoma cell line HepG2, as well as their responses to a model antiproliferative and cytotoxic agent, 5-fluorouracil. The complete protocol provides a simple and ready-to-use solution to initiate scaffold-free spheroid cultures in any laboratory with standard equipment for mammalian in vitro cell culture work. Thus, it allows to increase throughput and scale of spheroid culture experiments, which can be greatly utilized in different areas of biomedical, pharmaceutical and toxicological research.

Pro-Apoptotic Effect of Grape Seed Extract on MCF-7 Involves Transient Increase of Gap Junction Intercellular Communication and Cx43 Up-Regulation: A Mechanism of Chemoprevention.

Growing evidence suggests dietary antioxidants reduce the risk of several cancers. Grape seeds extracts (GSE) are a rich source of polyphenols known to have antioxidant, chemopreventive and anticancer properties. Herein, we investigated the in vitro effects and putative action mechanisms of a grape seed extract (GSE) on human breast cancer cells (MCF-7). The effects of GSE were evaluated on cell proliferation, apoptosis and gap-junction-mediated cell-cell communications (GJIC), as basal mechanism involved in the promotion stage of carcinogenesis. GSE (0.05-100 µg/mL) caused a significant dose- and time-dependent inhibition of MCF-7 viability and induced apoptotic cell death, as detected by Annexin-V/Propidium Iodide. Concurrently, GSE induced transient but significant enhancement of GJIC in non-communicating MCF-7 cells, as demonstrated by the scrape-loading/dye-transfer (SL/DT) assay and an early and dose-dependent re-localization of the connexin-43 (Cx43) proteins on plasma membranes, as assayed by immunocytochemistry. Finally, real-time-PCR has evidenced a significant increase in cx43 mRNA expression. The results support the hypothesis that the proliferation inhibition and pro-apoptotic effect of GSE against this breast cancer cell model are mediated by the GJIC improvement via re-localization of Cx43 proteins and up-regulation of cx43 gene, and provide further insight into the action mechanisms underlying the health-promoting action of dietary components.

Assessment of Hepatotoxic Potential of Cyanobacterial Toxins Using 3D In Vitro Model of Adult Human Liver Stem Cells.

Cyanotoxins microcystin-LR (MC-LR) and cylindrospermopsin (CYN) represent hazardous waterborne contaminants and potent human hepatotoxins. However, in vitro monolayer cultures of hepatic cell lines were found to recapitulate, poorly, major hepatocyte-specific functions and inadequately predict hepatotoxic effects of MC-LR and CYN. We utilized 3-dimensional (3D), scaffold-free spheroid cultures of human telomerase-immortalized adult liver stem cells HL1-hT1 to evaluate hepatotoxic potential of MC-LR and CYN. In monolayer cultures of HL1-hT1 cells, MC-LR did not induce cytotoxic effects (EC50 > 10 micromol/L), while CYN inhibited cell growth and viability (48h-96h EC50 ≈ 5.5-0.6 micromol/L). Growth and viability of small growing spheroids were inhibited by both cyanotoxins (≥0.1 micromol/L) and were associated with blebbing and disintegration at the spheroid surface. Hepatospheroid damage and viability reduction were observed also in large mature spheroids, with viability 96h-EC50 values being 0.04 micromol/L for MC-LR and 0.1 micromol/L for CYN, and No Observed Effect Concentrations <0.01 micromol/L. Spheroid cultures of adult human liver stem cells HL1-hT1 exhibit sensitivity comparable to cultures of primary hepatocytes and provide a simple, practical, and cost-effective tool, which can be effectively used in environmental and toxicological research, including assessment of hepatotoxic potential and effect-based monitoring of various samples contaminated with toxic cyanobacteria.

The Role of the Mitochondria in the Evolution of Stem Cells, Including MUSE Stem Cells and Their Biology.

From the transition of single-cell organisms to multicellularity of metazoans, evolutionary pressures selected new genes and phenotypes to cope with the oxygenation of the Earth's environment, especially via the symbiotic acquisition of the mitochondrial organelle. There were many new genes and phenotypes that appeared, namely, stem cells, low-oxygen-micro-environments to house these genes ("niches"), new epigenetic mechanisms to regulate , selectively, the gene repertoire to control proliferation, differentiation, apoptosis, senescence and DNA protection mechanisms, including antioxidant genes and DNA repair. This transition required a critical regulation of the metabolism of glucose to produce energy for both the stem cell quiescent state and the energy-requiring differentiated state. While the totipotent-, embryonic-, pluripotent-, and a few adult organ-specific stem cells were recognized, only relatively recently, because of the isolation of somatic cell nuclear transfer (SCNT) stem cells and "induced pluripotent stem" cells, challenges to the origin of these "iPS" cells have been made. The isolation and characterization of human MUSE stem cells and more adult organ-specific adult stem cells have indicated that these MUSE cells have many shared characteristics of the "iPS" cells, yet they do not form teratomas but can give rise to the trigeminal cell layers. While the MUSE cells are a subset of human fibroblastic cells, they have not been characterized, yet, for the mitochondrial metabolic genes, either in the stem cell state or during their differentiation processes. A description of other human adult stem cells will be made to set future studies of how the MUSE stem cells compare to all other stem cells.

Chemopreventive Agents Attenuate Rapid Inhibition of Gap Junctional Intercellular Communication Induced by Environmental Toxicants.

Altered gap junctional intercellular communication (GJIC) has been associated with chemical carcinogenesis, where both chemical tumor promoters and chemopreventive agents (CPAs) are known to conversely modulate GJIC. The aim of this study was to investigate whether attenuation of chemically inhibited GJIC represents a common outcome induced by different CPAs, which could be effectively evaluated using in vitro methods. Rat liver epithelial cells WB-F344 were pretreated with a CPA for either 30 min or 24 h, and then exposed to GJIC-inhibiting concentration of a selected tumor promoter or environmental toxicant [12-O-tetradecanoylphorbol-13-acetate (TPA), lindane, fluoranthene, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), perfluorooctanoic acid (PFOA), or pentachlorophenol]. Out of nine CPAs tested, quercetin and silibinin elicited the most pronounced effects, preventing the dysregulation of GJIC by all the GJIC inhibitors, but DDT. Metformin and curcumin attenuated the effects of three GJIC inhibitors, whereas the other CPAs prevented the effects of two (diallyl sulfide, emodin) or one (indole-3-carbinol, thymoquinone) GJIC inhibitor. Significant attenuation of chemically induced inhibition of GJIC was observed in 27 (50%) out of 54 possible combinations of nine CPAs and six GJIC inhibitors. Our data demonstrate that in vitro evaluation of GJIC can be used as an effective screening tool for identification of chemicals with potential chemopreventive activity.

Modulation by aspirin and naproxen of nucleotide alterations and tumors in the lung of mice exposed to environmental cigarette smoke since birth.

Chemoprevention provides an important strategy for cancer control in passive smokers. Due to the crucial role played by smoke-related chronic inflammation in lung carcinogenesis, of special interest are extensively used pharmacological agents, such as nonsteroidal anti-inflammatory drugs (NSAIDs). We evaluated the ability of aspirin and naproxen, inhibitors of both cyclooxygenase-1 and cyclooxygenase -2, to modulate environmental cigarette smoke (ECS)-induced lung carcinogenesis in A/J mice of both genders. Based on a subchronic toxicity study in 180 postweaning mice, we used 1600 mg/kg diet aspirin and 320 mg/kg diet naproxen. In the tumor chemoprevention study, using 320 mice, exposure to ECS started soon after birth and administration of NSAIDs started after weaning. At 10 weeks of life, the NSAIDs did not affect the presence of occult blood in feces. As assessed in a subset of 40 mice, bulky DNA adducts and 8-hydroxy-2'-deoxyguanosine levels were considerably increased in ECS-exposed mice and, irrespective of gender, both NSAIDs remarkably inhibited these nucleotide alterations. After exposure for 4 months followed by 5 months in filtered air, ECS induced a significant increase in the yield of surface lung tumors, the 43.7% of which were adenomas and the 56.3% were adenocarcinomas. Oct-4 (octamer-binding transcription factor 4), a marker of cell stemness, was detected in some adenocarcinoma cells. The NAIDs attenuated the yield of lung tumors, but prevention of ECS-induced lung adenomas was statistically significant only in female mice treated with aspirin, which supports a role for estrogens in ECS-related lung carcinogenesis and highlights the antiestrogenic properties of NSAIDs.

Oxidative stress-induced biomarkers for stem cell-based chemical screening.

Stem cells have been considered for their potential in pharmaceutical research, as well as for stem cell-based therapy for many diseases. Despite the potential for their use, the challenge remains to examine the safety and efficacy of stem cells for their use in therapies. Recently, oxidative stress has been strongly implicated in the functional regulation of cell behavior of stem cells. Therefore, development of rapid and sensitive biomarkers, related to oxidative stress is of growing importance in stem cell-based therapies for treating various diseases. Since stem cells have been implicated as targets for carcinogenesis and might be the origin of "cancer stem cells", understanding of how oxidative stress-induced signaling, known to be involved in the carcinogenic process could lead to potential screening of cancer chemopreventive and chemotherapeutic agents. An evaluation of antioxidant states reducing equivalents like GSH and superoxide dismutase (SOD), as well as reactive oxygen species (ROS) and nitric oxide (NO) generation, can be effective markers in stem cell-based therapies. In addition, oxidative adducts, such as 4-hydroxynonenal, can be reliable markers to detect cellular changes during self-renewal and differentiation of stem cells. This review highlights the biomarker development to monitor oxidative stress response for stem cell-based chemical screening.

The Concept of "Cancer Stem Cells" in the Context of Classic Carcinogenesis Hypotheses and Experimental Findings.

In this Commentary, the operational definition of cancer stem cells or cancer initiating cells includes the ability of certain cells, found in a heterogeneous mixture of cells within a tumor, which are able to sustain growth of that tumor. However, that concept of cancer stem cells does not resolve the age-old controversy of two opposing hypotheses of the origin of the cancer, namely the stem cell hypothesis versus the de-differentiation or re-programming hypothesis. Moreover, this cancer stem concept has to take into account classic experimental observations, techniques, and concepts, such as the multi-stage, multi-mechanism process of carcinogenesis; roles of mutagenic, cytotoxic and epigenetic mechanisms; the important differences between errors of DNA repair and errors of DNA replication in forming mutations; biomarkers of known characteristics of normal adult organ-specific stem cells and of cancer stem cells; and the characteristics of epigenetic mechanisms involved in the carcinogenic process. In addition, vague and misleading terms, such as carcinogens, immortal and normal cells have to be clarified in the context of current scientific facts. The ultimate integration of all of these historic factors to provide a current understanding of the origin and characteristics of a cancer stem cell, which is required for a rational strategy for prevention and therapy for cancer, does not follow a linear path. Lastly, it will be speculated that there exists evidence of two distinct types of cancer stem cells, one that has its origin in an organ-specific adult stem cell that is 'initiated' in the stem cell stage, expressing the Oct4A gene and not expressing any connexin gene or having functional gap junctional intercellular communication (GJIC). The other cancer stem cell is derived from a stem cell that is initiated early after the Oct4A gene is suppressed and the connexin gene is expressed, which starts early differentiation, but it is blocked from terminal differentiation.

A paradigm shift is required for the risk assessment of potential human health after exposure to low level chemical exposures: a response to the toxicity testing in the 21st century report.

Chemicals are known to be associated with birth defects, cancer, cardiovascular diseases, immunological, reproductive, and neurological disorders. In response to recent reviews of limitations of current concepts and techniques for toxicity testing, this commentary challenges the paradigm that chemicals are directly responsible for DNA damage in the genomic-nuclear DNA in relevant cells of the human body. This challenge is not that mutations do not play roles in human-inherited or somatic diseases but that chemical exposures bring about disease end points by epigenetic mechanisms or by alterations in adult stem cell numbers in utero (ie, the Barker hypothesis) or postnatally, by selecting preexisting mutated cells. Classic concepts, that is, multistage, multimechanism process of carcinogenesis, stem cell theory of cancer, and newer and ignored concepts, such as cancer stem cells and cell-cell communication, will be used to support the view that the toxic effect of chemicals is mediated by nonmutagenic mechanisms at human relevant exposures.

Actin and Vimentin proteins with N-terminal deletion detected in tumor-bearing rat livers induced by intraportal-vein injection of Ha-ras-transfected rat liver cells.

The introduction of the tumorigenic v-Ha-ras oncogene-transformed rat liver epithelial cells (WBras), which is deficient in gap junctional intercellular communication (GJIC), into F344 rats, induces significant formation of hepatocellular tumors. GJIC plays a major role in maintaining tissue homeostasis. Using this in vivo tumor model system, we used 2-dimensional electrophoresis with isoelectric focusing in the first dimension and SDS-PAGE in the second dimension to globally identify proteins that are uniquely expressed in the livers of WBras-treated rats as compared to the sham control. Immunoblotting was used to identify Ras and Connexin43, which were the positive and negative marker proteins, respectively, of the introduced WBras cells. As predicted, immunoblotting indicated that the whole liver of tumor-bearing animals exhibited a decreased level of Connexin43 and an increased level of Ras. Connexin43 and GJIC were expressed and functional in normal liver, but not in the tumor. In addition to these 2 markers, an additional 4 proteins exhibited decreased levels and 2 proteins exhibited increased levels in the livers of tumor-bearing animals. N-Terminal sequencing analysis was used to identify these proteins, which were glucose-regulated protein 78, 2 isoforms of heat shock protein 60, and the beta-chain of ATP synthase for the down regulated proteins, and beta-Actin with a 46 amino acid deletion from its N-terminus and Vimentin with a 71 amino acid deletion from its N-terminus for the up regulated proteins. These data offer potentially new markers of liver tumorigenicity, particularly, Vimentin. (

Cancer Prevention and Therapy of Two Types of Gap Junctional Intercellular Communication⁻Deficient "Cancer Stem Cell".

Early observations showed a lack of growth control and terminal differentiation with a lack of gap junctional intercellular communication (GJIC). Subsequent observations showed that epigenetic tumor promoters and activated oncogenes, which block gap junction function, provide insights into the multi-stage, multi-mechanism carcinogenic process. With the isolation of embryonic induced pluri-potent stem cells and organ-specific adult stem cells, gap junctions were linked to early development. While tumors and tumor cell lines are a heterogeneous mixture of "cancer stem cells" and "cancer non-stem cells", the cancer stem cells seem to be of two types, namely, they express (a) no connexin genes or (b) connexin genes, but do not have functional GJIC. These observations suggest that these "cancer stem cells" originate from normal adult stem cells or from the de-differentiation or re-programming of somatic differentiated cells. This "Concept Paper" provides a hypothesis that "cancer stem cells" either originate from (a) organ-specific adult stem cells before the expression of the connexin genes or (b) organ-specific adult stem cells that just express gap junction genes but that the connexin proteins are rendered dysfunctional by activated oncogenes. Therefore, cancer prevention and therapeutic strategies must account for these two different types of "cancer stem cell".

What Can Chemical Carcinogenesis Shed Light on the LNT Hypothesis in Radiation Carcinogenesis?

To protect the public's health from exposure to physical, chemical, and microbiological agents, it is important that any policy be based on rigorous scientifically based research. The concept of "linear no-threshold" (LNT) has been implemented to provide guideline exposures to these agents. The practical limitation to testing this hypothesis is to provide sufficient samples for experimental or epidemiological studies. While there is no universally accepted understanding of most human diseases, there seems to be better understanding of cancer that might help resolve the "LNT" model. The public's concern, after being exposed to radiation, is the potential of producing cancer. The most rigorous hypothesis of human carcinogenesis is the "multistage, multimechanism" chemical carcinogenesis model. The radiation carcinogenesis LNT model, rarely, if ever, built it into their support. It will be argued that this multistage, multimechanism model of carcinogenesis, involving the "initiation" of a single cell by a mutagen event, followed by chronic exposure to threshold levels of epigenetic agents or conditions that stimulate the clonal expansion of the "initiated" cell, can convert these benign cells to become invasive and metastatic. This "promotion" process can be interrupted, thereby preventing these initiated cells from transitioning to the "progression" process of invasion and metastasis.

Tumor promoting properties of a cigarette smoke prevalent polycyclic aromatic hydrocarbon as indicated by the inhibition of gap junctional intercellular communication via phosphatidylcholine-specific phospholipase C.

Inhibition of gap junctional intercellular communication (GJIC) and the activation of intracellular mitogenic pathways are common hallmarks of epithelial derived cancer cells. We previously determined that the 1-methyl and not the 2-methyl isomer of anthracene, which are prominent cigarette smoke components, activated extracellular receptor kinase, and inhibited GJIC in WB-F344 rat liver epithelial cells. Using these same cells, we show that an immediate upstream response to 1-methylanthracene was a rapid (<1 min) release of arachidonic acid. Inhibition of phosphatidylcholine-specific phospholipase C prevented the inhibition of GJIC by 1-methylanthracene. In contrast, inhibition of phosphatidylinositol specific phospholipase C, phospholipase A(2), diacylglycerol lipase, phospholipase D, protein kinase C, and tyrosine protein kinases had no effect on 1-methylanthracene-induced inhibition of GJIC. Inhibition of protein kinase A also prevented inhibition of GJIC by 1-methylanthracene. Direct measurement of phosphatidylcholine-specific phospholipase C and sphingomyelinase indicated that only phosphatidylcholine-specific phospholipase C was activated in response to 1-methylanthracene, while 2-methylanthracene had no effect. 1-methylanthracene also activated p38-mitogen activated protein kinase; however, like extracellular kinase, its activation was not involved in 1-methylanthracene-induced regulation of GJIC, and this activation was independent of phosphatidylcholine-specific phospholipase C. Although mitogen activated protein kinases were activated, Western blot analyzes indicated no change in connexin43 phosphorylation status. Our results indicate that phosphatidylcholine-specific phospholipase C is an important enzyme in the induction of a tumorigenic phenotype, namely the inhibition of GJIC; whereas mitogen activated protein kinases triggered in response to 1-methylanthracene, were not involved in the deregulation of GJIC.

3-Methylthiopropionic acid ethyl ester, isolated from Katsura-uri (Japanese pickling melon, Cucumis melo var. conomon), enhanced differentiation in human colon cancer cells.

The fully ripened fruit of Katsura-uri Japanese pickling melon ( Cucumis melo var. conomon) has rarely been used for food because the midripened fruit is utilized for making pickles, but the fully ripened fruit is no longer valuable for pickles due to the fruit body being too soft. We have considered the utilization of the fully ripened Katsura-uri fruit that may be used for nonpickling products, particularly if the fully ripened fruit demonstrated health benefits such as anticarcinogenic properties. The phytochemical extract from the fully ripened fruit of Katsura-uri Japanese pickling melon was purified via a bioassay-guided fractionation scheme, which was based on the induction of differentiation in a RCM-1 human colon cancer cell line. On the criteria of two differentiation markers (duct formation and alkaline phosphatase activity), the most potent fraction contained a compound identified as 3-methylthiopropionic acid ethyl ester, based on GC retention time, EI-MS, (1)H NMR, and (13)C NMR spectra. Previously, the role of 3-methylthiopropionic acid ethyl ester was considered as an odor producing compound in many fruits, but this study indicates potential medical benefits of this compound.

Commentary: "re-programming or selecting adult stem cells?".

The recent observations that embryonic stemness-associated genes could assist in the "de-differentiation" of adult skin fibroblast cells to "embryonic-like stem cells", using the "somatic cell nuclear transfer" techniques, have been interpreted as indicating a "re-programming" of genes. These reports have demonstrated a "proof of principle" approach to by-pass many, but not all, of the ethical, scientific and medical limitations of the "therapeutic cloning" of embryonic stem cells from embryos. However, while the interpretation that real "re-programming" of all those somatic fibroblastic differentiation genes might be correct, there does exists an alternative hypothesis of these exciting results. Based on the fact that multipotent adult stem cells exist in most, if not all, adult organs, the possibility exists that all these recent "re-programming" results, using the somatic nuclear transfer techniques, actually were the results of transferred rare nuclear material from the adult stem cells residing in the skin of the mouse, monkey and human samples. An examination of the rationale for this challenging hypothesis has been drawn from the hypothesis of the "stem cell theory of cancer", as well as from the field of human adult stem cells research.

Protective role of connexin 32 in steady-state hematopoiesis, regeneration state, and leukemogenesis.

The role of gap junctions formed by connexins (Cxs) has been implicated in the homeostatic regulation of multicellular systems. Primitive hematopoietic progenitor cells form a multicellular system, but a previous report states that Cx32 is not expressed in the bone marrow. Thus, a question arises as to why Cx molecules are not detected in the hematopoietic tissue other than in stromal cells. Based on our preliminary study, which suggested a potential impairment of hematopoiesis in Cx32-knockout (KO) mice, the objectives of the present study were to determine whether Cx32 functions in the bone marrow during steady-state hematopoiesis and to examine its possible protective roles during regeneration after chemical abrasions and during leukemogenesis after the administration of a secondary genotoxic chemical, methyl nitrosourea (MNU). As a result, the Cx32 molecule, functioning in the hematopoietic stem cell (HSC) compartment during steady-state hematopoiesis, was observed for the first time; the expressions of Cx32 at the mRNA level, as determined by polymerase chain reaction analysis, and at the protein level, determined using an anti-Cx32 antibody, were observed only in the lin(-)c-kit(+) HSC fraction, using a combination of immunobead-density gradient and immunomagnetic bead separation. Hematopoiesis was impaired in the absence of Cx32, and it was delayed during regeneration after chemical abrasion with 5-fluorouracil at 150 mg/kg body wt in Cx32-KO mice. Cx32-KO mice showed increased leukemogenicity compared with wild-type mice after MNU injection; furthermore, in a competitive assay for leukemogenicity in mice that had been lethally irradiated and repopulated with a mixed population of bone marrow cells from Cx32-KO mice and wild-type mice, the resulting leukemias originated predominantly from Cx32-KO bone marrow cells. In summary, the role of Cx32 in hematopoiesis was not previously recognized, and Cx32 was expressed only in HSCs and their progenitor cells. The results indicate that Cx32 in wild-type mice protects HSCs from chemical abrasion and leukemogenic impacts.