James E. Trosko

Inhibition of gap junctional intercellular communication in human teratocarcinoma cells by organochlorine pesticides

Inhibition of intercellular communication, as measured by metabolic cooperation between 6-thioguanine-sensitive and 6-thioguanine-resistant Chinese hamster V79 cells, has been previously shown to be correlated with a large variety of known tumor promoters, including some of the organochlorine pesticides. Since further evidence concerning the effects of those known or suspected animal tumor promoters on human cells is needed, three organochlorine pesticides, dieldrin, aldrin, and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), were tested for their ability to inhibit metabolic cooperation between 6-thioguanine-sensitive (6TGs, HTP3-4) and 6-thioguanine-resistant (6TGr, HTXTG-1) human teratocarcinoma cells. Similar to the effect of the known mouse skin tumor promoter 12-tetradecanoyl phorbol-13-acetate (TPA), all three pesticides inhibited gap junctional intercellular communication within a noncytotoxic dose range. The dose-response curves of these chemicals were similar to those of other known tumor promoters on Chinese hamster V79 cells. In addition, the transfer of [3H]uridine between teratocarcinoma cells in contact was reduced after pesticide treatment.

Scrape-loading and dye transfer: A rapid and simple technique to study gap junctional intercellular communication

Gap junction-mediated intercellular communication has been recognized in cells from different tissues of various organisms and has been implicated in a variety of cellular functions and dysfunctions. Here we describe a new, direct and rapid technique with which to study this cellular phenomenon. It employs scrape-loading to introduce a low molecular weight (MW) fluorescent dye, Lucifer yellow CH (MW 457.2) into cells in culture and allows the monitoring of its transfer into contiguous cells. In communication-competent cells the dye transmission occurred within minutes after loading. The involvement of membrane junctions in Lucifer yellow transfer was verified by the concurrent loading of a high MW marker dye conjugate, rhodamine dextran (MW 10,000). Once introduced intracellularly the rhodamine dextran is unable to cross the relatively narrow membrane junctions. Chemicals of variable potency known to block junctional communication were tested in Chinese hamster V79 cells and other mammalian cells. The results showed effective blockage of the dye transfer at non-cytotoxic doses. This new technique can be applied to a wide variety of mammalian (including human) cells. In addition, it has the potential to be utilized as a rapid screening assay to detect chemicals that can modulate intercellular communication and to study their mechanism of action.

Radiation risk to low fluences of particles may be greater than we thought

Based principally on the cancer incidence found in survivors of the atomic bombs dropped in Hiroshima and Nagasaki, the International Commission on Radiation Protection (ICRP) and the United States National Council on Radiation Protection and Measurements (NCRP) have recommended that estimates of cancer risk for low dose exposure be extrapolated from higher doses by using a linear, no-threshold model. This recommendation is based on the dogma that the DNA of the nucleus is the main target for radiation-induced genotoxicity and, as fewer cells are directly damaged, the deleterious effects of radiation proportionally decline. In this paper, we used a precision microbeam to target an exact fraction (either 100% or ≤20%) of the cells in a confluent population and irradiated their nuclei with exactly one α particle each. We found that the frequencies of induced mutations and chromosomal changes in populations where some known fractions of nuclei were hit are consistent with non-hit cells contributing significantly to the response. In fact, irradiation of 10% of a confluent mammalian cell population with a single α particle per cell results in a mutant yield similar to that observed when all of the cells in the population are irradiated. This effect was significantly eliminated in cells pretreated with a 1 mM dose of octanol, which inhibits gap junction-mediated intercellular communication, or in cells carrying a dominant negative connexin 43 vector. The data imply that the relevant target for radiation mutagenesis is larger than an individual cell and suggest a need to reconsider the validity of the linear extrapolation in making risk estimates for low dose, high linear-energy-transfer (LET) radiation exposure.

Absence of excision of ultraviolet-induced cyclobutane dimers in xeroderma pigmentosum.

XERODERMA pigmentosum is a rare human skin disease which is inherited as an autosomal recessive mutation and has been known by dermatologists for nearly a century [ 1-31. The major clinical symptom is high actinic skin carcinogenesis [ 1.4.51. There are two forms of the disease: one shows only the skin symptoms, the other more uncommon form shows additional neurological symptoms and is known as the de Sanctis Cacchione syndrome [5-71. Whether the neurological symptoms should be regarded as part of xeroderma pigmentosum or due to separate genetic factors is as yet unknown [2,5-71. Fibroblasts[& 91 and epithelial cells[lO] from the skin in both forms of this disease appear to perform low or negligible amounts of DNA repair replication after irradiation with ultraviolet (u.v.) light, but the level of such residual amounts cannot yet be correlated with the two forms of the disease since the number of cases studied thus far is too small[& 111. Previous studies on DNA repair in de Sanctis Cacchione skin fibroblasts suggested that an early step in DNA repair involving enzymatic strand breakage is defective [9,12]. Such an hypothesis has also been advanced by Setlow et al. [ 131 for skin fibroblasts from the non-neurological form of xeroderma pigmentosum. They showed that thymine-containing dimers are excised after U.V. damage of normals cells but not of xeroderma cells. Since the two forms of the disease might show some biochemical differences, we have now investigated the excision of thyminecontaining dimers in cultured cells from patients with the de Sanctis Cacchione syndrome. Preliminary results of this investigation have already been reported [9, 121. Sterile 1 mm punch biopsies were taken from apparently normal regions of the arms of three de Sanctis Cacchione patients and two normal persons. Fibroblast cultures were developed in Eagles minimum essential medium with 3 mglml dextrose, non-essential amino acids, and 15 percent fetal calf serum. Usually, 1-5-2 months elapsed between the time of biopsy and the start of any experiments with fibroblast cultures. They were then used throughout the period of steady growth until cultures degenerated[ 14, IS]. Human embryonic fibroblasts (WI-38) were also used and these

Chemical, Oncogene and Growth Factor Inhibition of Gap Junctional Intercellular Communication: An Integrative Hypothesis of Carcinogenesis

Most, if not all, cancer cells have some dysfunction in gap-junction-mediated intercellular communication, either because of defects in cell adhesion or inability to have functional gap junctional communication. In addition, most, if not all, tumor-promoting chemicals and conditions down-regulate gap junction function, while some antitumor-promoting chemicals can up-regulate gap junctional communication. Several oncogenes are associated with down-regulation of gap junction function and several hormone and growth regulators, known to be tumor promoters, are also able to down-regulate gap junction function. On the other hand, some tumor suppressor genes have been linked to the up-regulation of gap junctions. Based on these observations, it is hypothesized that, if a progenitor cell is unable to perform gap junctional intercellular communication, normal growth control and cell differentiation would not be possible, thereby favoring the development of malignant neoplasia.

Inhibition of gap junctional intercellular communication by perfluorinated fatty acids is dependent on the chain length of the fluorinated tail

Perfluorinated fatty acids (PFFAs), such as perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA), are known peroxisome proliferators and hepatocarcinogens. A causal link between an increase in the oxidative stress by peroxisomes and tumor promotion has been proposed to explain the hepatocarcinogenicity of PFOA and PFDA. However, the down‐regulation of gap junctional intercellular communication (GJIC) has also been linked to the tumor‐promoting properties of many carcinogens. Therefore, the effect of PFFAs on GJIC in WB‐rat liver epithelial cells was determined. The chain length of the PFFAs tested for an effect on GJIC ranged from 2 to 10, 16 and 18 carbons. Carbon lengths of 7 to 10 inhibited GJIC in a dose–response fashion, whereas carbon lengths of 2 to 5, 16 and 18 did not appreciably inhibit GJIC. Inhibition occurred within 15 min and was reversible, with total recovery from inhibition occurring within 30 min after the removal of the compound from the growth medium. This short time of inhibition suggests that GJIC was modified at the post‐translational level. Also, this short time period was not long enough for peroxisome proliferation. The post‐translational modification of the gap junction proteins was not a consequence of altered phosphorylation as determined by Western blot analysis. Perfluorooctanesulfonic acid also inhibited GJIC in a dose–response fashion similar to PFDA, indicating that the determining factor of inhibition was probably the fluorinated tail, which required 7–10 carbons. Our results suggest that PFFAs could potentially act as hepatocarcinogens at the level of gap junctions in addition to or instead of through peroxisome proliferation.Int. J. Cancer 78:491–495, 1998.

Inhibition of connexin43 gap junctional intercellular communication by TPA requires ERK activation

The phorbol ester, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), is a potent inhibitor of gap junctional intercellular communication (GJIC). This inhibition requires activation of protein kinase C (PKC), but the events downstream of this kinase are not known. Since PKC can activate extracellular signal regulated kinases (ERKs) and these also downregulate GJIC, we hypothesized that the inhibition of GJIC by TPA involved ERKs. TPA treatment (10 ng/ml for 30 min) of WB‐F344 rat liver epithelial cells strongly activated p42 and p44 ERK‐1 and ‐2, blocked gap junction‐mediated fluorescent dye‐coupling, and induced connexin43 hyperphosphorylation and gap junction internalization. These effects were completely prevented by inhibitors of PKC (bis‐indolylmaleimide I; 2 μM) and ERK activation (U‐0126; 10 μM). These data suggest that ERKs are activated by PKC in response to TPA treatment and are downstream mediators of the gap junction effects of the phorbol ester. J. Cell. Biochem. 83: 163–169, 2001.

Ignored Hallmarks of Carcinogenesis: Stem Cells and Cell‐Cell Communication

Abstract: Hanahan and Weinberg (2000, Cell 100: 57‐70) listed “hallmarks” of cancer that must be considered in order to understand the underlying determinants of carcinogenesis: (a) self‐sufficiency in growth signals; (b) insensitivity to growth‐inhibitory (antigrowth) signals; (c) evasion of programmed cell death (apoptosis); (d) limitedless replicative potential; (e) sustained angiogenesis; and (f) tissue invasion and metastasis. While these are important phenotypic markers, important concepts—the role of pluripotent stem cells and gap junctional intercellular communication (GJIC)—must be brought into this analysis of carcinogenesis. Carcinogenesis is a multistage, multimechanism process consisting of a single cell that has been irreversibly blocked from terminal differentiation (the initiation stage). The promotion phase is a potentially reversible or interruptible clonal expansion of the initiated cell by a combination of growth stimulation and inhibition of apoptosis. When the expanded initiated cells accrue sufficient mutations and epigenetic alterations to become growth stimulus independent and resistant to growth inhibitors and apoptosis, to have unlimited replicative potential and invasive and metastatic phenotypes, then the progression phase has been achieved. The hypothesis that integrates these hallmarks is that the stem cell and its early progenitor cell are the target cells for the initiation event. These cells are naturally immortal and become mortal only when they are induced to terminally differentiate and lose their telomerase activity. These two types of initiated cells are suppressed by either secreted negative growth regulators (the stem cells) or GJIC (the early initiated progenitor cells). Promoters inhibit either the secreted growth inhibitor to initiated stem cells or GJIC between the initiated progenitor cells and the normal progenitor cells. When a stable resistance to the secreted negative growth regulator or permanent downregulation of GJIC has occurred, the cell has entered the progression phase. These two new concepts contradict the current paradigm that the first phase of carcinogenesis is the immortalization of a normal cell followed by its neoplastic transformation. Our hypothesis is that the first stage of carcinogenesis must prevent the “mortalization” or terminal differentiation of a naturally immortal cell. Chemoprevention and chemotherapeutic implications suggest that one must induce connexin genes in initiated stem cells and restore GJIC in initiated early progenitor cells.

From Adult Stem Cells to Cancer Stem Cells

Abstract:  Carcinogenesis is characterized by “initiation,”“promotion,” and “progression” phases. The “stem cell theory” and “de‐differentiation” theories are used to explain the origin of cancer. Growth control for stem cells, which lack functional gap junctional intercellular communication (GJIC), involves negative soluble or niche factors, while for progenitor cells, it involves GJIC. Tumor promoters, hormones, and growth factors inhibit GJIC reversibly. Oncogenes stably inhibit GJIC. Cancer cells, which lack growth control and the ability to terminally differentiate and to apoptose, lack GJIC. The Oct3/4 gene, a POU (Pit‐Oct‐Unc) family of transcription factors was thought to be expressed only in embryonic stem cells and in tumor cells. With the availability of normal adult human stem cells, tests for the expression of Oct3/4 gene and the stem cell theory in human carcinogenesis became possible. Human breast, liver, pancreas, kidney, mesenchyme, and gastric stem cells, HeLa and MCF‐7 cells, and canine tumors were tested with antibodies and polymerase chain reaction (PCR) primers for Oct3/4. Adult human breast stem cells, immortalized nontumorigenic and tumor cell lines, but not the normal differentiated cells, expressed Oct3/4. Adult human differentiated cells lose their Oct‐4 expression. Oct3/4 is expressed in a few cells found in the basal layer of human skin epidermis. The data demonstrate that normal adult stem cells and cancer stem cells maintain expression of Oct3/4, consistent with the stem cell hypothesis of carcinogenesis. These Oct‐4 positive cells might represent the “cancer stem cells.” A strategy to target “cancer stem cells” is to suppress the Oct‐4 gene in order to cause the cells to differentiate.

Metformin represses self-renewal of the human breast carcinoma stem cells via inhibition of estrogen receptor-mediated OCT4 expression.

Metformin, a Type II diabetic treatment drug, which inhibits transcription of gluconeogenesis genes, has recently been shown to lower the risk of some diabetes-related tumors, including breast cancer. Recently, “cancer stem cells” have been demonstrated to sustain the growth of tumors and are resistant to therapy. To test the hypothesis that metformin might be reducing the risk to breast cancers, the human breast carcinoma cell line, MCF-7, grown in 3-dimensional mammospheres which represent human breast cancer stem cell population, were treated with various known and suspected breast cancer chemicals with and without non-cytotoxic concentrations of metformin. Using OCT4 expression as a marker for the cancer stem cells, the number and size were measured in these cells. Results demonstrated that TCDD (100 nM) and bisphenol A (10 µM) increased the number and size of the mammospheres, as did estrogen (10 nM E2). By monitoring a cancer stem cell marker, OCT4, the stimulation by these chemicals was correlated with the increased expression of OCT4. On the other hand, metformin at 1 and 10 mM concentration dramatically reduced the size and number of mammospheres. Results also demonstrated the metformin reduced the expression of OCT4 in E2 & TCDD mammospheres but not in the bisphenol A mammospheres, suggesting different mechanisms of action of the bisphenol A on human breast carcinoma cells. In addition, these results support the use of 3-dimensional human breast cancer stem cells as a means to screen for potential human breast tumor promoters and breast chemopreventive and chemotherapeutic agents.