Differential effects of TPA and pristane on gene expression and transformation in mouse epidermal cells.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) or 2,6,10,14-tetramethylpentadecane (pristane) on gene expression and transformation were examined using two clones (P+, TPA transformation sensitive and P-, TPA resistant) of the mouse epidermal cell line JB6. Results from transformation studies indicated pristane was more efficient, i.e., lower concentrations were required to elicit an equivalent response, in transforming the P+, but not the P-, clone of JB6 compared to TPA. Furthermore, results from these studies demonstrated either TPA or pristane was effective in the transactivation of the chloramphenicol acetyltransferase gene under the regulatory control of most viral promoter/enhancer elements transfected into the P+, but not the P-, clone of JB6. However, if a consensus cAMP response element was linked to the simian virus 40 early promoter, pristane activation was observed in both P+ and P- cells. The differential effects of these two compounds suggest that while they have similar characteristics, they may utilize different pathways to elicit their effects.

Interaction of HPV-18 and nitrosomethylurea in the induction of squamous cell carcinoma.

A strong correlation exists between the presence of specific types of human papillomavirus (HPV) and the development of anogenital cancer, as well as significant epidemiologic evidence suggesting smokers are at increased risk of developing cervical, vulvar and/or anal carcinomas. Primary and human papillomavirus type 18 (HPV-8)-immortalized human keratinocytes were used to address the co-carcinogenic potential of HPV and nitrosomethylurea (NMU) in tumorigenesis. Only cells containing HPV-18 and treated with NMU and the tumor-promoting phorbol ester, TPA, were transformed to a malignant phenotype. An in vitro system is described which initiates studies involving the mechanisms of HPV and chemical carcinogen co-operation in the etiology of squamous cell carcinomas.

Alteration of the DCC tumor-suppressor gene in tumorigenic HPV-18 immortalized human keratinocytes transformed by nitrosomethylurea.

A human papillomavirus type 18 (HPV-18)-immortalized human keratinocyte cell line (1811) has been transformed to tumorigenicity in nude mice by treatment with the carcinogen nitrosomethylurea (NMU). The NMU transformants (1811-NMU-T) showed additional chromosome alterations as compared with parental 1811 cells, including 18q deletion in two of two 1811-NMU-T lines analysed. Restriction fragment length polymorphism (RFLP) analysis indicated that both 1811-NMU-T lines had lost one allele of the 18q deleted in colon cancer (DCC) tumor-suppressor gene. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that DCC expression was absent or barely detectable in the 1811-NMU-T cells as compared with 1811 or normal keratinocytes, suggesting that the remaining DCC allele in the 1811-NMU-T cells was also altered. These studies indicate that reduction or loss of DCC expression may be an important step in NMU transformation of HPV-immortalized cells to tumorigenicity.

Pristane induced gene activation.

Studies were performed to examine the effects of 2,6,10,14-tetramethyl pentadecane (pristane) versus 12-O-tetradecanoylphorbol 13-acetate (TPA) on the activation of the CAT gene under the regulatory control of viral promoter/enhancer elements transfected into NIH-3T3, CV-1 and COS-7 cells. The results of these studies demonstrated that (1) pristane or TPA induced trans-activation of SV2cat, HIVcat, RSVcat and MMTVcat in cells transfected with each respective plasmid construct, (2) only pristane induced activation of pA10cat and pOSP/11 and (3) neither TPA nor pristane trans-activated pSV0cat. Furthermore, treatment with either pristane or TPA elicited changes in the morphology of each of the cell lines. Collectively these results indicate that pristane is a potent inducer of gene expression and exhibits similar characteristics as the tumor promoter, TPA.

Increased intracellular calcium is associated with progression of HPV-18 immortalized human keratinocytes to tumorigenicity.

Studies were conducted using normal and human papillomavirus Type 18 (HPV-18) immortalized human keratinocytes to assess possible alterations in the differentiation process as a consequence of increased intracellular calcium concentration. Normal keratinocytes exposed to increased extracellular calcium or the phorbol ester TPA, exhibited terminal differentiation characteristics. However, late passage HPV-18 immortalized keratinocytes (designated FEP-1811) were resistant to such terminal differentiation signals. Flow cytometric analyses of 1811 cells at various stages of passage in culture revealed progressively higher levels of intracellular calcium in the immortalized cells with passage in culture when compared to normal, primary keratinocytes. Furthermore, 1811 cells isolated from tumors which developed in irradiated nude mice contained the highest level of intracellular calcium of all the cells examined. These results suggest that an increase in the concentration of intracellular calcium is associated with progression of HPV-18 immortalized keratinocytes to tumorigenicity.

Pristane induced effects on chromatin of rat lymphoid cells.

The effects of pristane on the conformation of chromatin in cells isolated from the lymphoid tissues of pristane-treated Copenhagen rats were examined by flow cytometry, thermal denaturation, sensitivity to enzymatic digestion, and histone protein analyses. Decreases were observed in the fluorescent intensities of propidium iodide (PI) stained nuclei isolated from lymphoid cells of pristane-treated rats when compared with normal rat lymphoid nuclei. Studies to address the possible basis for the pristane-induced changes in the DNA staining characteristics of lymphocytes demonstrated that 1) there were no decreases in the amount of DNA present in the nuclei, 2) nuclei isolated from pristane treated rats were less sensitive to thermal denaturation, as well as DNase I enzymatic digestion, and 3) there were apparent increases in the expression of the H1 histone proteins. Collectively, these results suggest that pristane elicits a conformational change in the chromatin which may be mediated by altered expression of nuclear-associated histone proteins.

Thymic tumors induced by 3-methylcholanthrene treatment of rat Peyer's patches.

Studies were performed to characterize thymic tumors which were induced after a single injection of 500 microgram or 3-methylcholanthrene (3-MC) into surgically exposed Peyer's patches (PP) of Copenhagen rats. Detailed gross, histological, and morphological analyses revealed thymic tumors differing in size and weight (1 to greater than 8 g) with distorted architecture and infiltration by lymphocytes and epithelial cells in varying proportions. Approximately 25% of the rats with thymic tumors exhibited abnormal spleens, whereas 66% developed low grade leukemias. A majority of the thymic tumors contained cells which exhibited (1) phenotypic markers characteristic of normal thymocytes, (2) abnormal DNA, and (3) increased percentages in S + G2 phases of the cell cycle. Further studies of tumor cell isolates demonstrated an increased frequency of colony formation on soft agar, as well as the ability to elicit thymic tumors upon transplantation. Collectively these studies describe chemically induced thymic lymphomas.

The role of the Peyer's patch in the carcinogenesis of lymphoid cells.

The dose response to 3-methylcholanthrene (3-MC), the promoter effects of 2,6,10,14-tetramethylpentadecane (pristane) and the target-organ specificity in the preferential induction of B-lymphoid malignancies versus thymic tumors were examined. Lymphoid malignancies were induced in approximately 30% of the Copenhagen rats treated with injections in Peyer's patches (PP) of low, intermediate or high doses of 3-MC. A low dose of 3-MC induced B-lymphocytic leukemias or B lymphomas, whereas thymic tumors were detected in rats treated with high doses. Co-treatment of rats with pristane and 3-MC resulted in increased incidences and decreased latency of the lymphoid malignancies observed, suggesting that pristane acts as a tumor promoter. To address the possible role of PP in the induction events, PP were surgically removed after 3-MC treatment and the remaining small intestine anastomosed. Thymic tumors, but no B-lymphoid malignancies, were observed, indicating that the PP environment was important in the induction of the B-lymphoid malignancies. Radiotracer studies also revealed that appreciable amounts of 3-MC were disseminated to the thymus within 24 hr after treatment of PP with a high dose of 3-MC. Furthermore, direct intrathymic injection of the thymus with 3-MC resulted in the development of thymic tumors only. These results support the hypothesis that the PP has an important role in early events in the carcinogenesis of B lymphocytes and in the dissemination of 3-MC to the thymus.

Pristane induced changes in rat lymphocyte membrane fluidity.

The ability of pristane (2,6,10,14-tetramethylpentadecane) to act as a membrane perturbant was examined. Data obtained from rats treated with pristane by either intraperitoneal injection or the diet indicated there were significant increases over normal in the amount of pristane in lymphoid cells; 50-89% was incorporated into the plasma membranes. Fluorescence polarization analyses, using 1,6-diphenyl-1,3,5-hexatriene, of normal plasma membrane isolates demonstrated that splenic and Peyer's patch lymphocytic membranes were more viscous than those of the thymus, mesenteric lymph nodes or peripheral blood. Studies to assess the effects of pristane on membrane viscosity demonstrated that there were significant differences in the viscosities of plasma membrane isolates from lymphocytes of normal versus pristane treated rats. The observed changes were dependent on route of administration, length of exposure and the lymphoid organ examined.

Dietary effects of pristane on rat lymphoid tissues.

Studies were conducted to assess the normal tissue-associated levels of pristane (2,6,10,14,-tetramethylpentadecane) in Copenhagen rats during ontogeny and adult life and to address whether or not dietary pristane can be adsorbed from the gut and disseminated throughout the body. During the course of this study the possible effects of dietary pristane on chromatin conformation of lymphoid cells were also examined by flow cytometry. The data indicated that 1) pristane crossed the placenta and accumulated in fetal tissues, 2) neonates were exposed to pristane via the colostrum, 3) there were significant increases in the amount of tissue-associated pristane in young adults and subsequent redistribution of the pristane to the muscle and adipose tissues in older rats and 4) after dietary exposure, significantly elevated levels of pristane were associated with the tissues and concomitant changes in chromatin conformation were observed. Collectively, these results suggest that pristane was adsorbed from dietary sources, disseminated to the tissues and exerted a transient, yet marked effect on chromatin of lymphoid cells in rats.

Conformational changes in the DNA of hybridoma cells from pristane treated mice.

The effects of pristane on the DNA of hybridoma cells propagated as ascitic tumors in pristane-primed BALB/c mice were determined using flow cytometric analyses. Hybridoma cells maintained in vitro or cell isolates from solid tumors which developed in unprimed mice injected with hybridoma cells exhibited similar propidium iodide (PI) staining characteristics. In contrast, PI stained cells isolated from ascites which developed in pristane-primed mice injected with the hybridoma cells displayed significant decreases in fluorescence intensity. Diphenylamine studies and analyses of pH 10 treated cells indicated that the actual DNA content of the hybridoma cells was not altered by exposure to pristane. Furthermore, the altered staining characteristics of the ascitic tumor cells were reversible in that the fluorescence intensity after serial in vitro passage of the ascites cells was similar to that of the parent cell line which had not been exposed to pristane. In addition, there was a direct correlation between the altered PI staining characteristics and the presence of cell-associated pristane as determined by gas-liquid chromatography analyses of cell extracts. Collectively these results suggest that pristane may have a direct effect on the DNA conformation of hybridoma cells which may in turn enhance their growth as ascitic tumors. The possible role of such an altered DNA conformation in hybridoma cells on the in vivo development of ascites is discussed.

Changes in the DNA of lymphocytes from pristane treated rats.

The effects of pristane (2,6,10,14-tetramethylpentadecane) on the cellular DNA of lymphoid cells from Copenhagen rats were examined by flow cytometry. Significant reductions in the mean relative fluorescent intensities of propidium iodide (PI) stained lymphocytes from peripheral blood, spleen, thymus and lymph nodes were observed after a single intraperitoneal injection of pristane. The altered PI staining characteristics were observed as early as 4 days and reached a maximum decrease between 1-4 weeks (depending upon the lymphoid cells examined) post pristane treatment. The pristane-induced effects on peripheral blood lymphocytes were observed to be dose dependent, transient and reinducible by a subsequent exposure to pristane. Further analyses, using gas-liquid chromatography to detect pristane in the blood and lymphoid tissues of treated rats, indicated significant increases over normal amounts of pristane. Furthermore, correlations existed between the times of maximum decrease in the fluorescence of PI stained cells and the amounts of pristane detected within the respective lymphoid tissues. By contrast no changes in the PI staining characteristics of kidney cells were observed, even though appreciable amounts of pristane were detected in this organ. Diphenylamine analyses indicated no differences in the amounts of DNA in lymphoid cells from pristane treated and untreated rats. Furthermore, lymphocytes from pristane-treated rats did not exhibit decreased fluorescence when fixed at pH 10 rather than pH 7.4 prior to PI staining. Collectively these results suggest that pristane may preferentially induce qualitative rather than quantitative changes in the DNA of lymphocytes.

The role of the Peyer's patch in carcinogenesis. II. 3-Methylcholanthrene-induced lymphoid malignancies in rat Peyer's patches.

To determine whether B or T lymphoid malignancies could be induced following the exposure of lymphoid cells within different lymphoid organs to a potential chemical carcinogen, 3-methylcholanthrene (3-MC) was directly injected into surgically exposed Peyer's patches (PP), mesenteric lymph nodes (MLN), axillary lymph nodes (ALN) or spleens (SP) of Copenhagen rats. A high incidence of lymphoproliferative disorders was observed within rats which received 3-MC injections into the PP, but not in MLN, ALN or SP injected groups of rats. In addition to the PP environment, the dose of 3-MC and exposure to pristane were important factors in the induction of T versus B cell disorders. Whereas the B cell diseases were observed in pristane-treated rats which also received PP injections of low doses (5 or 50 micrograms) of 3-MC, in animals receiving a higher dose (500 micrograms) a much higher incidence of T cell disorders was detected. The observed lymphoproliferative diseases were categorized as B lymphocytic leukemias, B lymphomas or thymic lymphomas on the basis of histological examination of the tissues, white blood cell numbers and differentials, and the morphological and phenotypic characteristics of cell isolates. Abnormal DNA staining characteristics, increased soft agar cloning frequencies and metastasis of the leukemia or tumor cells indicated that malignant cells were associated with the proliferative diseases. Collectively the data indicate that primary lymphoid malignancies of either B or T cell origins may be preferentially and reproducibly induced by localizing low or high doses respectively, of 3-MC within the PP of rats exposed to pristane. These results suggest that PP may have a possible role in the carcinogenesis of lymphocytes following the exposure to chemical carcinogens via the gastrointestinal tract.