Alteration in the retinoblastoma gene associated with immortalization of human fibroblasts treated with60Co gamma rays

Genetic analysis was carried out in human fibroblasts (KMST-6) immortalized by treatment with⁶⁰Co gamma rays in order to determine if any genetic change was involved in the immortal transformation of human cells. Analysis by restriction fragment length polymorphism revealed an alteration in chromosome 13q12–14, in which the retinoblastoma (RB) gene locus (13q14) is located. Then the RB gene itself was examined. Structural abnormalities in the RB gene were detected by Southern blot analysis. Furthermore, abnormal RB protein (pRB) was expressed in immortalized KMST-6 cells, as shown by in vitro phosphorylation, whereas normal KMS-6 cells expressed the intact pRB. These findings indicated that inactivation of the RB gene is one of the key events of the immortalization of human cells.Abbreviations RB retinoblastoma - pRB retinoblastoma gene product (protein) - T simian virus 40 large T antigen - E1A adenovirus E1A protein     

Effect of Okadaic Acid, a Protein Phosphatase Inhibitor, on Heat Stress-Induced HSP72 Synthesis and Thermotolerance

Heat stress proteins (HSPs), in particular HSP72, seem to play a major role in cell protection against lethal stresses such as hyperthermia or ischemia. HSP synthesis is negatively regulated by protein phosphatases, which are implicated in dephosphorylation processes. In the present study, we have investigated the effect of okadaic acid (OA, a protein phosphatase inhibitor) on heat stress-induced HSP72 synthesis and thermotolerance in smooth muscle cells (SMC).SMC were heat stressed (42°C for 20 minutes) in the presence of 250 nM OA (HS+OA cells) or its vehicle (HS+V cells). Control (OA or V) cells were not heat stressed. HSP72 mRNA expression was determined 1, 1.5, 3, and 6 hours after heat stress by RT-PCR, and HSP72 synthesis was determined 6, 12, 24, 48, and 72 hours after heat stress by Western blotting. SMC survival of lethal hyperthermia (47°C for 90 minutes) was assessed 6, 24, and 48 hours after heat stress by a tetrazolium assay.The maximal expression of HSP72 mRNA was markedly prolonged in HS+OA cells (until 6 hours after heat stress) compared to HS+V cells (1 hour after heat stress). The kinetics of HSP72 synthesis and thermotolerance of SMC were not different between HS+OA and HS+V cells. Baseline HSP72 mRNA and protein expression were similar in control V and OA cells.In conclusion, okadaic acid treatment of SMC potentiated HSP72 mRNA expression without affecting heat stress-induced HSP72 synthesis and thermotolerance.     

Transformation of rodent immortalized and embryo cells by oncogenes. I. Mouse (NIH 3T3) and rat (FR 3T3) immortalized fibroblasts

Immortalized mouse NIH 3T3 cells were transformed by gene transfer of DNA isolated from a human bladder tumor cell line and plasmids containing an activated human Ha-ras oncogene insert. For gene transfer the calcium-phosphate co-precipitation method was used. Transformation was evaluated by morphological focus formation, growth in soft agar and tumor development in nude mice. In addition, immortalized rat FR 3T3 cells were transformed by Ha-ras, too. The co-transfer of ras and myc oncogenes did not enhance focus formation in FR 3T3 cells.     

Comparison of major cytoskeletons among normal human fibroblasts, immortal human fibroblasts transformed by exposure to Co-60 gamma rays, and the latter cells made tumorigenic by treatment with Harvey murine sarcoma virus

Immortally transformed human fibroblasts in general acquire an epithelial shape, while normal human fibroblasts demonstrate a spindle-shaped feature. In order to investigate this difference, three types of major cytoskeletal elements, namely, F-actin, tubulin, and vimentin of immortal human fibroblasts were morphologically compared with those of normal human fibroblasts. As a result, a significant difference was observed in the distribution and in the number of F-actin fibers between immortal and normal fibroblasts. The cells of three immortally transformed fibroblast lines, KMST-6, WI-38 VA-13, and SUSM-1, showed a striking reduction in the number, and an altered pattern of organization, of actin fibers. On the other hand, in the normal fibroblasts, actin fibers ran parallel to each other along the long axis of the cells. Tubulin and vimentin showed no significant difference between the immortal and normal cells. Our present data show that the morphological changes seen in the immortally transformed cells are due to the disorganization and the decrease in number of actin fibers. Interestingly, both the immortal cells (KMST-6), which were not tumorigenic, and the Harvey murine sarcoma virus-transformed KMST-6 cells, which were tumorigenic and demonstrated an enhanced expression of the ras gene, revealed an overall similarity in the organization pattern and the number of actin fibers. These findings seem to indicate that the immortally transformed cells have already acquired some cancer characteristics.     

Effect of okadaic acid and calyculin-A, two protein phosphatase inhibitors, on thyrotropin-stimulated triiodothyronine secretion in cultured sheep thyroid cells

We have studied the effect of two protein phosphatase inhibitors on thyrotropin (TSH)-stimulated triiodothyronine (T₃) production by sheep thyroid cells grown in primary culture. Incubation of sheep thyrocytes with okadaic acid (OA) and calyculin-A (CL-A), two potent inhibitors of type 1 (PP1) and type 2A (PP2A) protein phosphatases, resulted in an increase of TSH-stimulated T₃ production. This effect was detected using concentrations as low as 0.1 pM with OA and 1 fM with CL-A. An inhibitory effect on T₃ production, due to cellular death, was observed with 6 nM OA and 1 nM CL-A. In the absence of TSH, OA or CL-A had no effect on T₃ production by thyrocytes. Forskoline (10 μM), an activator of adenylate cyclase, increased the basal and TSH-stimulated T₃ release by sheep thyroid cells; this effect was increased by OA in cells grown in the basal state but not in the presence of TSH. These results suggest that the marine toxins OA and CL-A, two potent inhibitors of PP-1 and PP-2A, have significant stimulatory effects on T₃ secretion promoted by TSH and FK. These observations indicate that these proteins could be important mediators of thyroid hormone production.     

Okadaic acid regulation of the retinoblastoma gene product is correlated with the inhibition of growth factor-induced cell proliferation in mouse fibroblasts.

Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, was used to study the mechanism of action of transforming growth factor beta (TGF-beta) on cell cycle progression in C3H/10T1/2 mouse embryonic fibroblasts, where TGF-beta exerts a growth-stimulatory effect. Concentrations of okadaic acid as low as 5 nM inhibited TGF-beta (5 ng/ml)- or 10% serum-induced [3H]thymidine incorporation into postconfluent, quiescent cells. Further, these inhibitory effects were observed when okadaic acid was added as late as 10 hr after TGF-beta or serum stimulation. Since C3H/10T1/2 fibroblasts undergo the G1/S transition at 10-14 hr after TGF-beta and 8-12 hr after serum stimulation, these observations indicate that a phosphatase activity may be required for S-phase entry. In a parallel experiment, okadaic acid partially inhibited TGF-beta-induced [14C]leucine incorporation by 20-65%, depending upon the okadaic acid concentration. In conjunction with the effect of okadaic acid on DNA and protein synthesis, Western blot analysis indicated that okadaic acid inhibited phosphorylation of the retinoblastoma gene product and decreased its protein level, even when added 10 hr after TGF-beta or 8 hr after serum stimulation. These findings strongly suggest that protein phosphatases play a pivotal role for S-phase entry in mouse fibroblasts. Moreover, protein phosphatases may be required in the intermediate steps of TGF-beta or serum growth factor signal-transduction pathways for the stimulation of phosphorylation of the retinoblastoma protein, especially in late G1.     

Inhibition of cell growth by a hypothalamic peptide.

A fraction purified from acetic acid extracts of porcine hypothalami was found to contain significant antimitogenic activity when tested in normal and neoplastic cell lines. Addition of this hypothalamic material (1-100 micrograms/ml) to culture media significantly inhibited [3H]thymidine incorporation into cellular DNA in several cell lines. Amino acid incorporation into pituitary proteins and uridine incorporation into RNA were also significantly reduced by this factor(s). Addition to the culture media of this hypothalamic material at 5 micrograms/ml and 50 micrograms/ml per day decreased by 17% and 36%, respectively, cell numbers of 3T6 fibroblast cell cultures. Time-response curves showed that the inhibition of [3H]thymidine incorporation into DNA in 3T6 fibroblast cells begins within 2 hr after adding this fraction to the culture medium. The inhibitory action cannot be explained by a direct cytotoxic effect since 3T6 cells labeled with 51Cr and incubated for 6 hr in the presence of this hypothalamic fraction fail to show an increase in the release of 51Cr into the medium as compared with controls. Incubation with trypsin and chymotrypsin completely abolished the antimitogenic activity of this material and pepsin decreased it. This strongly suggests that the antimitogenic activity exhibited by this fraction is due to a polypeptide(s). These observations provide evidence for the presence in the mammalian hypothalamus of an antimitogenic peptide(s) that may be involved in the regulation of cell proliferation.     

Flat reversion by okadaic acid of raf and ret-II transformants.

Okadaic acid is a non-phorbol 12-myristate 13-acetate (PMA)-type tumor promoter on mouse skin and known to be a potent inhibitor of serine/threonine protein phosphatases. Contrary to expectation from its tumor-promoting activity, okadaic acid was shown to have a potential to revert the phenotypes of cells transformed by raf and ret-II to that of normal cells. Two to 3 days after addition of 8 ng of okadaic acid per ml to the culture medium, raf and ret-II transformants changed to flat cells and gained contact inhibition. The amount of fibronectin, which was decreased in malignant transformed cells, was increased in the flat revertants. Moreover, okadaic acid caused a dose-dependent loss of ability to grow in soft agar. The morphology of the cells reverted to malignant phenotype within 1 week after removal of okadaic acid. The levels of mRNA and protein of activated c-raf in flat revertants were similar to those in parental transformed cells. The level of mRNA of ret-II was also not changed by flat reversion. No induction of flat reversion was observed with okadaic acid tetramethyl ether, an inactive compound, or a phorbol ester, PMA. As okadaic acid is a potent inhibitor of protein phosphatases 1 and 2A, the possibility that these phosphatases are involved in signal transduction from the raf and ret-II oncogenes is suggested.     

Insulin-like growth factor-I and -II in the ovary of a bony fish, Oreochromis mossambicus, the tilapia: in situ hybridisation, immunohistochemical localisation, Northern blot and cDNA sequences

In FRTL-5 cells, cultured over a period of more than 3 years, different properties of the cells have been observed to undergo spontaneous changes in the course of aging, i.e. after an increase in the number of passages. This consists mainly in alterations in their morphological phenotype and in some of their functional properties. The morphology of the cells displayed a progressive disruption of the monolayer organization with a loss of cell–cell contacts and a marked rounding-up of the cells. The uptake of iodide was not modified nor was the expression of thyroglobulin (Tg) mRNA as determined at various time intervals in the course of the cells culturing. Estimation of the proliferation by counting the frequency of [³H]thymidine labeled nuclei revealed an age-related decline in the sensitivity to TSH mitogenic action associated with a reciprocal increase in the insulin synergistic effect. Aged cells (±40 passages) lost their apoptosis sensitivity to the phosphatase inhibitor, okadaic acid (OA) but not to cycloheximide (CHX) and/or actinomycin D (act. D) exposure. Altogether these observations favor the existence of a shift towards transformed properties with only partial loss of differentiated functions.     

Neoplastic development: paradoxical relation between impaired cell growth at low population density and excessive growth at high density.

The role of heritable, population-wide cell damage in neoplastic development was studied in the 28 L subline of NIH 3T3 cells. These cells differ from the 17(3c) subline used previously for such studies in their lower frequency of "spontaneous" transformation at high population density and their greater capacity to produce large, dense transformed foci. Three cultures of the 28 L subline of NIH 3T3 cells were held under the constraint of confluence for 5 wk (5 wk 1 degree assay) and then assayed twice in succession (2 degrees and 3 degrees assays) for transformed foci and saturation density. After the 2 degrees assay, the cells were also passaged at low density to determine their exponential growth rates and cloned to determine the size and morphological features of the colonies. Concurrent measurements were made in each case with control cells that had been kept only in frequent low-density passages and cells that had been kept at confluence for only 2 wk (2 wk 1 degree). Two of the three cultures transferred from the 2 degrees assay of the 5 wk 1 degree cultures produced light transformed foci, and the third produced dense foci. The light focus-forming cultures grew to twice the control saturation density in their 2 degrees assay and 6-8 times the control density in the 3 degrees assay; saturation densities for the dense focus formers were about 10 times the control values in both assays. All three of the cultures transferred from the 2 degrees assay of the 5 wk 1 degree cultures multiplied at lower rates than controls at low densities, but the dense focus formers multiplied faster than the light focus formers. The reduced rates of multiplication of the light focus formers persisted for > 50 generations of exponential multiplication at low densities. Isolated colonies formed from single cells of the light focus formers were of a lower population density than controls; colonies formed by the dense focus formers were slightly denser than the controls but occupied only half the area. A much higher proportion of the colonies from the 5 wk 1 degree cultures than the controls consisted of giant cells or mixtures of giant and normal-appearing cells. The results reinforce the previous conclusion that the early increases in saturation density and light focus formation are associated with, and perhaps caused by, heritable, population-wide damage to cells that is essentially epigenetic in nature. The more advanced transformation characterized by large increases in saturation density and dense focus formation could have originated from rare genetic changes, such as chromosome rearrangements, known to occur at an elevated frequency in cells destabilized by antecedent cellular damage.     

Transformation of human umbilical cord blood T cells by human T-cell leukemia/lymphoma virus.

Several isolates of human T-cell leukemia/lymphoma virus (HTLV) were transmitted to normal human T cells obtained from the umbilical cord blood of newborns. T cells from seven specimens were immortalized by infection with different HTLV isolates and their properties were compared with those of activated uninfected normal T cells grown in the presence of T-cell growth factor (TCGF) and with those of HTLV-positive neoplastic T-cell lines derived from patients with T-cell malignancies. The HTLV-infected cells generally belonged to a class of mature T cells (OKT4+ and Leu 3A+) and differed from the normal uninfected cells in that they could be propagated in culture indefinitely; possessed altered morphology, including convoluted nuclei and some bi- and multinucleated giant cells; formed large clumps in culture; demonstrated a diminished requirement for TCGF; had an increased density of TCGF receptors; often became completely independent of exogenous TCGF; and expressed HLA-DR determinants. These properties of the HTLV-infected cord blood T cells contrasted to those of uncultured cord blood T cells and of cord blood cells stimulated with mitogen and grown with TCGF but resembled the characteristics of T-cell lines established previously from patients with HTLV-associated T-cell malignancies. This in vitro system offers a unique opportunity to study the basic mechanism involved in abnormal growth and neoplastic transformation of a specific class of human T cells.     

Endocrine, biochemical, and morphological studies of a pituitary adenoma secreting growth hormone, thyrotropin (TSH), and alpha-subunit: evidence for secretion of TSH with increased bioactivity

A 40-yr-old man who had acromegaly and hyperthyroidism due to a GH/TSH-secreting pituitary adenoma is described. Serum free T4 was 2.8 ng/dl, free T3 was 1.1 ng/dl, and TSH was 1.2-1.5 microU/ml; the latter was measured in an immunoradiometric assay with a sensitivity of 0.07 microU/ml. Serum TSH was immunologically identical to standard TSH and did not decrease during a T3 suppression test. Serum free alpha-subunit and the molar alpha-subunit to TSH ratio were high (6.1 ng/ml and 31.2, respectively). TRH administration induced significant increases in both GH (+129%) and alpha-subunit (+156%) levels. Conversely, dopamine infusion resulted in a decrease in serum GH (-66%) and alpha-subunit (-43%) levels, and subsequent administration of the dopamine antagonist sulpiride induced significant increases in both GH and alpha-subunit (+393% and +106%, respectively). Similarly, somatostatin infusion inhibited GH (-43%) and alpha-subunit (-61%) secretion. Serum TSH levels were not affected by TRH, dopamine, or somatostatin. The biological to immunological activity ratio of serum TSH purified by immunoaffinity chromatography and measured in an adenylate cyclase assay was significantly increased compared to that in serum from hypothyroid or euthyroid subjects [biological to immunological activity ratio, 6.9 +/- 0.2 (+/- SD) vs. 4.4 +/- 1.1; P less than 0.001]. In gel chromatography, the apparent mol wt of the patient's TSH was smaller than that of the controls. After adenomectomy, all of the altered parameters of pituitary function became normal. Double gold particle immunostaining of the adenomatous tissue showed that all of the cells contained secretory granules positive for GH and alpha-subunit, while very few cells were positive for TSH beta as well as GH and alpha-subunit. These data indicate that in this patient serum TSH had an apparent mol wt smaller than that of normal TSH and an increased biological activity which, along with the autonomous TSH secretion, account for hyperthyroidism in the presence of low normal TSH levels; alpha-subunit originated from the same adenomatous cells that secreted GH but not TSH, thus explaining the in vivo observation that alpha-subunit responses to several agents were dissociated from TSH responses and parallel to GH responses; and TSH and GH were colocalized in a minority of the neoplastic cells.     

Relationship between O6-alkylguanine-DNA alkyltransferase activity and N-methyl-N''-nitro-N-nitrosoguanidine-induced mutation, transformation, and cytotoxicity in C3H/10T1/2 cells expressing exogenous alkyltransferase genes.

While a great deal of evidence has directly implicated the importance of O6-alkylation of guanine in the mutagenicity of alkylating agents, evidence demonstrating the oncogenic potential of this lesion has been largely indirect. We have combined a well-studied in vitro neoplastic transformation system (using C3H/10T1/2 mouse cells) with a proven method of gene transfection for expressing the bacterial O6-alkylguanine-DNA alkyltransferase (AT; EC 2.1.1.63) repair genes ada and ogt to generate subclones which possess augmented repair capability toward specific DNA lesions. The products of these genes specifically and differentially repair O6-methylguanine (O6-MeGua), O4-methylthymine (O4-MeThy), and methylphosphotriesters. We show that the level of expression of either the ada or the ogt AT gene in C3H/10T1/2 cells directly correlates with protection against mutation to ouabain resistance by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Subclones expressing 70 fmol of AT per 10(6) cells exhibited a mutation frequency approximately 1/40th of that of clones expressing 15 fmol of AT per 10(6) cells when treated with MNNG at 0.4 micrograms/ml. Protection against mutagenesis by MNNG at 0.8 micrograms/ml, however, did not exceed 12-fold even in subclones expressing greater than 100 fmol of AT per 10(6) cells. As an MNNG dose of 0.6 micrograms/ml was sufficient to saturate more than 95% of the AT activity in any of the clones, the residual mutation frequency may have been caused by unrepaired O6MeGua lesions. In contrast to mutagenesis, protection against neoplastic transformation in vitro, in cells expressing high levels of AT, was most pronounced in cells treated with the highest dose of MNNG used (1.2 micrograms/ml). Low levels of transformation caused by MNNG at 0.4 and 0.8 micrograms/ml were not consistently inhibited in those clones. These data suggest that O6-MeGua formation is of major but not unique significance in the neoplastic transformation of C3H/10T1/2 cells by MNNG.     

Cytochemically demonstrable B-glucuronidase activity in normal and neoplastic human lymphoid cells

Mononuclear cell suspensions were prepared from 40 normal peripheral blood and lymphoid tissue specimens and 42 neoplastic specimens obtained from patients with malignant lymphoma and lymphocytic leukemia. These suspensions were analyzed for la antigens, surface immunoglobulin (Slg), sheep erythrocyte (E) rosette formation and, in some instances, acid alpha-naphthyl acetate esterase (ANAE) activity. The results of these studies were correlated with the expression of cytochemically demonstrable BG activity. The percentage of BG+ lymphocytes was found to be comparable, within 10%, to the percentage of E+ (T) cells in the majority of normal, non-neoplastic peripheral blood, tonsil, spleen, and lymph node specimens examined. Occasionally, the percentage of E+ cells exceeded the percentage of BG+ cells by 20% or more, suggesting the presence of an E+BG- T cell subpopulation. BG+ B lymphocytes were only demonstrated in 1 of 40 non-neoplastic lymphoid specimens. The neoplastic B cells in each of 14 B cell (la+Slg+E-) lymphomas were BG-. However, a variable proportion of the neoplastic cells isolated from 6 cases of B cell chronic lymphocytic leukemia and neoplastic plasma cells isolated from 7 cases of multiple myeloma expressed BG activity. Thus, it appears that both normal and neoplastic BG- and BG+ B lymphocyte populations exist; the latter may be related to a state of activation or a stage of B cell differentiation. The neoplastic cells isolated from 4 T cell (la-Slg-E+) malignancies were BG+ while those isolated from 3 T cell malignancies were BG-. The variable expression of BG activity by T cell malignancies may be related to T cell differentiation. Investigation of BG expression by T cell derived malignancies may prove useful in sorting out T cell phenotypes.     

Effects of retinoic acid on NIH3T3 cell transformation by the H-ras oncogene

Summary Exposure of NIH3T3 cells to retinoic acid resulted in a dose-dependent modulation of transformed focus formation after transfection with an activated H-ras oncogene. Inhibition induced by 10 M retinoic acid was maximal at 21.4% of control values. Maximal inhibition of transformation was found after exposure to 10 M retinoic acid between days 0 and 3 of the transfection period. This concentration was also inhibitory for colony formation upon transfection of the non-transforming geneaph, suggesting that retinoic acid acts primarily on the process of transfection to inhibit focus or colony formation. Exposure to retinoic acid during the late period of the transfection protocol (days 14–20) resulted in alterations in focus morphology. A transformed cell line containing H-ras underwent reversion of the transformed phenotype after 4 weeks of treatment with retinoic acid, as determined by alterations in cell morphology and anchorage-independent growth. Phenotypic reversion was not associated with changes in the expression of the exogenous H-ras or endogenous c-myc or c-fos oncogenes.Abbreviations DMEM Dulbecco's modified Eagle medium - SSC saline sodium citrate - TGF transforming growth factor This investigation was supported by grants CA 52925, CA 13343 and ES 00260     

Role for interleukin-6 and insulin-like growth factor-I via PI3-K/Akt pathway in the proliferation of CD56- and CD56+ multiple myeloma cells

OBJECTIVES: Several studies including ours have suggested that lack of CD56 in multiple myeloma (MM) defines a unique patient subset with poorer prognosis. However, the mechanism underlying this aggressive behavior of CD56(-) MM has not been well elucidated. Interleukin-6 (IL-6) or insulin-like growth factor I (IGF-I) induce proliferation of MM cells. In this study, we report about the relationship between CD56 expression and responsiveness to these cytokines. METHODS: We sorted out both CD56(-) and CD56(+) fractions from MM cell lines such as KMS-21-BM and U-266, and investigated their different responsiveness to IL-6 or IGF-I. Furthermore, we compared the effects of these cytokines on the regulation of cell-cycle distribution between CD56(-) and CD56(+) cells. RESULTS: Although CD56(-) cells in both KMS-21-BM and U-266 cells responded significantly to IL-6, CD56(+) cells did not. Ki-67(+) cells in the CD56(-) cells were significantly increased by IL-6. Western blotting showed that IL-6 phosphorylated Akt, and upregulated and downregulated the level of cyclin D1 and p27 protein in the CD56(-) KMS-21-BM cells, respectively. LY-294002 completely blocked these effects of IL-6. On the other hand, Ki-67(+) cells in the CD56(+) cells did not respond to IL-6. Anti-IGF-I mAb significantly reduced Ki-67(+) cells only in the CD56(+) cells. IGF-I phosphorylated Akt and upregulated cyclin D1 in the CD56(+) KMS-21-BM cells, which was completely blocked by LY294002. CONCLUSIONS: These results suggest that CD56(-) and CD56(+) MM cells could be stimulated by IL-6 and IGF-I, respectively, via PI3-K/Akt pathway, and provide useful information for anticytokine therapies.     

The regulation of apoptosis by activin and transforming growth factor-beta in early neoplastic and tumorigenic ovarian surface epithelium

Most ovarian neoplasms arise from the ovarian surface epithelium (OSE), and multiple growth factors have been implicated to influence the transformation from OSE. The present study was performed to investigate the role of activin and transforming growth factor-beta (TGFbeta) in normal and neoplastic OSE cells. An immortalized OSE cell line (IOSE-29) was generated from normal OSE by transfecting simian virus 40 large T antigen and was rendered tumorigenic after subsequent transfection with the E-cadherin gene (IOSE-29EC). The activin/inhibin subunits and activin receptors were expressed at both messenger ribonucleic acids and protein levels in these cells, suggesting that activin may have an autocrine role in neoplastic OSE cells. Treatments with activin (1-100 ng/mL) resulted in a significant decrease in cell proliferation in both IOSE-29 and IOSE-29EC cells, although we have shown that it stimulated the growth of ovarian cancer cells and had no effect on normal OSE. This inhibitory effect was attenuated with cotreatment with follistatin. Treatment with TGFbeta (0.1-10 ng/mL) also significantly decreased the proliferation of normal, IOSE-29, and IOSE-29EC cells in a dose-dependent manner. In addition, treatments with both activin and TGFbeta resulted in an increase in DNA fragmentation in IOSE-29EC cells in a dose-dependent manner. This apoptotic effect of activin was attenuated by cotreatment with follistatin. Treatment with TGFbeta (1 and 10 ng/mL) resulted in a significant decrease in Bcl-2 protein (up to 50%) in IOSE-29EC, whereas no difference was observed in Bax protein levels. Therefore, down-regulated Bcl-2 by TGFbeta may eventually induce apoptosis in IOSE-29EC cells. In contrast, no difference was observed in Bax and Bcl-2 protein expression after treatment with activin. In conclusion, the present study indicates that activin and TGFbeta inhibited growth and induced apoptosis in early neoplastic (IOSE-29) and tumorigenic OSE (IOSE-29EC) cells. Furthermore, antiapoptotic Bcl-2 protein was down-regulated by TGFbeta, whereas no difference was produced in Bax protein by activin or TGFbeta treatment or in Bcl-2 protein by activin. These results suggest that activin and TGFbeta may play a role in growth inhibition and induction of apoptosis in early neoplastic and tumorigenic stage of ovarian cancer.     

The role of protein kinases and phosphatases in erythroid colony formation of elderly

Using H-7, HA1001, FK506, cyclosporin A (CsA) and okadaic acid (OA), which are protein kinase and phosphatase inhibitors, we examined qualitative changes in hematopoietic precursor cells due to aging from the viewpoint of the role of protein kinases and phosphatases. Though H-7 and OA suppressed erythroid colony formation both in the elderly (age: 72-92, median: 86) and the young (age: 22-39, median: 29), no change due to aging was noted. HA1001 did not affect erythroid colony formation either in the elderly or the young. Erythroid colony formation was enhanced by FK506 and CsA in the young, however, erythroid colony formation was suppressed in the elderly. Similar examinations using cell fractions of non-T, non-macrophage, non-T + T, and CD34 positive cells were performed in both groups. Enhancement of erythroid colony formation in the young and suppression in the elderly by FK506 using unseparated MNC disappeared after removal of T cells. Enhancement of colony formation in the young and suppression of colony formation in the elderly were recovered when T cells were added again. The effects of FK506 and CsA on erythroid colony formation were thought to be the results of T cell inactivation, and the different sensitivity to FK506 and CsA in the elderly and young seemed to be the result of changes in the control mechanisms of hematopoiesis, such as the regulation of cytokine production by T cells, caused by aging.     

G-protein-coupled receptor genes as protooncogenes: constitutively activating mutation of the alpha 1B-adrenergic receptor enhances mitogenesis and tumorigenicity.

The alpha 1B-adrenergic receptor (alpha 1B-ADR) is a member of the G-protein-coupled family of transmembrane receptors. When transfected into Rat-1 and NIH 3T3 fibroblasts, this receptor induces focus formation in an agonist-dependent manner. Focus-derived, transformed fibroblasts exhibit high levels of functional alpha 1B-ADR expression, demonstrate a catecholamine-induced enhancement in the rate of cellular proliferation, and are tumorigenic when injected into nude mice. Induction of neoplastic transformation by the alpha 1B-ADR, therefore, identifies this normal cellular gene as a protooncogene. Mutational alteration of this receptor can lead to activation of this protooncogene, resulting in an enhanced ability of agonist to induce focus formation with a decreased latency and quantitative increase in transformed foci. In contrast to cells expressing the wild-type alpha 1B-ADR, focus formation in "oncomutant"-expressing cell lines appears constitutively activated with the generation of foci in unstimulated cells. Further, these cell lines exhibit near-maximal rates of proliferation even in the absence of catecholamine supplementation. They also demonstrate an enhanced ability for tumor generation in nude mice with a decreased period of latency compared with cells expressing the wild-type receptor. Thus, the alpha 1B-ADR gene can, when overexpressed and activated, function as an oncogene inducing neoplastic transformation. Mutational alteration of this receptor gene can result in the activation of this protooncogene, enhancing its oncogenic potential. These findings suggest that analogous spontaneously occurring mutations in this class of receptor proteins could play a key role in the induction or progression of neoplastic transformation and atherosclerosis.     

Factors that affect human hemopoiesis are produced by T-cell growth factor dependent and independent cultured T-cell leukemia-lymphoma cells

Some laboratory results and clinical situations suggest that human T cells may be important in the regulation of growth of hematopoietic cells. Since the discovery of T-cell growth factor (TCGF), systems are now available for the long-term specific in vitro propagation of mature normal or neoplastic human T cells, providing an opportunity to study the influence of T cells on hematopoiesis. Recently, 24 cell lines from patients with cutaneous T-cell lymphoma (CTCL) and T-cell acute lymphoblastic leukemia (T-ALL) were grown with TCGF and then assessed for release of humoral factors that affect hematopoiesis. Conditioned media (CM) from these cell lines were tested for erythroid burst- promoting activity (BPA) and granulocyte colony-stimulating activity (CSA). BPA was detected in CM from 3/6 cultures of T-ALL patients and 4/6 CTCL cultures. CSA was found in the CM from 6/8 cultures of T-ALL patients, 7/12 CTCL cultures, and 3/4 CTCL cell lines that become independent of exogenous TCGF for growth. The CSA from several of the neoplastic T-cell cultures stimulated high levels of eosinophil colonies, a possible source of the eosinophilia seen in these patients. The ability of continuously proliferating human T lymphocytes, which retain functional specificity and responsiveness to normal humoral regulation, to produce factors that directly or indirectly stimulate myeloid and erythroid colony formation lends further credence to the role of T lymphocytes in regulating hematopoiesis.