Differential effects by Mad and Max on transformation by cellular and viral oncoproteins.

c-Myc is an essential component of the regulatory mechanisms controlling cell growth. Max is the obligatory partner of c-Myc for all its biological functions analysed to date. Recently two Max interacting proteins, Mad and Mxi1, have been identified. It has been suggested that these two proteins modulate c-Myc function, in the simplest model by competing with c-Myc for the interaction with Max. We have analysed different aspects of Mad function in comparison to Max. Native Mad/Max heterodimers bound specifically to a c-Myc/Max consensus DNA binding site. Furthermore Mad inhibited efficiently c-Myc, mutant p53, adenovirus E1a, or human papilloma virus type 16 transformation of rat embryo cells in cooperation with activated Ha-Ras. Myc transformed clones showed an increased cell cycle time and a reduced immortalization frequency after cotransfection with either mad or max. In contrast to Mad, Max did not inhibit E1a/Ha-Ras cotransformation but repressed c-Myc/Ha-Ras transformation efficiently. Mad delta N, an N-terminal deletion mutant of Mad, was as efficient in repressing c-Myc/Ha-Ras cotransformation as full length Mad but showed little inhibitory activity when assayed on E1a/Ha-Ras. Unlike wt Mad, Mad delta N had little effect on cell growth. Our data suggest that Mad affects cell growth at least in part by a c-Myc independent mechanism.

YY1 can inhibit c-Myc function through a mechanism requiring DNA binding of YY1 but neither its transactivation domain nor direct interaction with c-Myc.

The proto-oncoprotein c-Myc and the multifunctional transcriptional regulator YY1 have been shown previously to interact directly in a manner that excludes Max from the complex (Shrivastava et al., 1993). As binding to Max is necessary for all known c-Myc activities we have analysed the influence of YY1 on c-Myc function. We demonstrate that YY1 is a potent inhibitor of c-Myc transforming activity. The region in YY1 required for inhibition corresponds to a functional DNA-binding domain and is distinct from the domains necessary for direct binding to c-Myc. Furthermore the transactivation domain of YY1 was not necessary suggesting that gene regulation by YY1, for example through DNA bending or displacement of regulators from DNA, could be the cause for the negative regulation of c-Myc. This model of indirect regulation of c-Myc by YY1 was supported by the finding that although YY1 did not bind to the c-Myc transactivation domain (TAD) in vitro it was able to inhibit transactivation by Gal4-MycTAD fusion proteins in transient transfections. As for the inhibition of transformation, an intact DNA-binding domain of YY1 was necessary and sufficient for this effect. In addition YY1 did not alter c-Myc/Max DNA binding, further supporting an indirect mode of action. Our findings point to a role of YY1 as a negative regulator of cell growth with a possible involvement in tumor suppression.

Elevated expression of an exogenous c-myc gene is insufficient for transformation and tumorigenic conversion of established fibroblasts.

Two established rat fibroblast lines, differing only by their number of generations in culture, show dramatically different responses to the elevated c-myc expression delivered by an efficient murine c-myc retrovirus vector. Thus, a late passage (60 generation) FR3T3 line acquires a transformed and tumorigenic phenotype upon introduction of this activated c-myc gene as indicated by its altered morphology, high efficiency of focus formation, soft agar clonability, saturation density in monolayer culture, and short latency of tumorigenicity in syngeneic hosts. Remarkably, none of these characteristics, except for an increased refractility in monolayers and an epidermal growth factor (EGF)-dependent agar clonability, were observed in a variety of early passage (10 generation) FR3T3 c-myc clones. BALB/c A31 fibroblasts transfected with this c-myc retroviral vector behaved essentially the same as the FR3T3 early line except for their inability to grow in suspension in response to EGF. However, transformation and tumorigenic conversion of each of these three fibroblast lines was achieved by an activated ras oncogene. Hence, elevated c-myc expression is insufficient for transformation of established fibroblasts but depends upon other acquired cooperating functions which are not necessary for ras induced transformation. We also demonstrate that endogenous c-myc expression remains unaffected even in clones expressing a 100-fold excess of exogenous c-myc RNAs demonstrating that c-myc autoregulation is not operative in these cells.

Non-selective analysis of the transformation of FR3T3 rat cells by bovine papillomavirus type 1: regulations of viral transcription associated with phenotypic transformation.

Drug-resistant clones selected from FR3T3 rat cells after transfer of neo-BPV1 (Bovine Papillomavirus Type 1) DNA constructs became phenotypically transformed (focal transformation, growth in suspension and tumor formation) soon after selection (approximately 5 generations in culture). A frameshift mutation in ORF E5 abolished transformation, but did not prevent the autonomous maintenance of the DNA construct. A more complex situation was observed when the E2 transactivating function was abrogated. A minority of the E2(-)-neor clones became phenotypically transformed shortly after drug selection, but the majority maintained normal growth properties for 30 to 50 generations. The rate of viral transcription was uniformly high in cells which exhibited transformed growth properties early after selection (the E2- minority class and all the wild type transformants) and low in phenotypically normal cells (the majority of the E2- lines). The same low transcriptional activity and delayed expression of transformed growth properties had been observed after transfection of a similar construct carrying a wild type viral early region (69-T fragment), but lacking the late region. The elevated rate of viral transcription, which correlates with the immediate expression of transformation, appears therefore to require at least two distinct elements, the E2 transactivator function and sequences in the late region of the viral genome. In their absence, high transcription rates and transformation could be established only in a minority of the transfected clones, by an unknown, E2-independent mechanism. Evidence was obtained for a third transformation route which, in the absence of either E2 or the late region, led to the focal occurrence of transformed derivatives after 30 to 50 generations of normal growth, but was not associated with an overall increase in viral expression.

Cdc25A phosphatase suppresses apoptosis induced by serum deprivation.

The phosphatase Cdc25A was shown to be a target of the transcription factor c-Myc. Myc-induced apoptosis appeared dependent on Cdc25A expression and Cdc25A over-expression could substitute for Myc-triggered apoptosis. These findings suggested that an important downstream component of Myc-mediated apoptosis was identified. However and in contrast, we recently reported that during TNFalpha-induced apoptosis, which required c-Myc function, Cdc25A was down-regulated in a human carcinoma cell line. We now provide evidence that Cdc25A rendered the non-transformed rat embryonic cell line 423 refractory to apoptosis, which was induced by serum deprivation and in absence of detectable c-myc levels. The survival promoting activity of cdc25A was abolished upon infection of cells with a full-length cdc25A antisense construct. To identify the signaling proteins mediating the survival function of the phosphatase, cdc25A- and akt- over-expressing pooled clones were exposed to selected chemicals, which inhibit or activate key proteins in signaling pathways. Inhibition of apoptosis by SU4984, NF023 and Rapamycin placed Cdc25A and Akt function downstream of FGF.R, PDGF.R, and compensated G-protein- and PP2A- activity. Interestingly, upon treatment with LY-294002, cdc25A- and akt- over-expressing clones exhibited similar apoptotic patterns as control cells, which indicates that neither Akt- nor Cdc25A-mediated survival functions are dependent on PI.3 kinase activity in rat 423 cells. In cdc25A-overexpressing cells increased levels of serine 473 phosphorylated Akt were found, which co-precipitated with Cdc25A and Raf1. Since activation of proteins requires dephosphorylation of particular residues in addition to site-specific phosphorylation, the anti-apoptotic effect of Cdc25A might derive from its participation in a multimeric protein complex with phosphoAkt and Raf1, two prominent components of survival pathways.

Subcellular localisation of Cdc25A determines cell fate.

Cell division cycle 25A (Cdc25A) was shown to colocalise both with nuclear and cytoplasmic proteins. Recently, we have demonstrated that overexpressed Cdc25A promoted the survival of rat 423 cells through indirect activation of PKB-protein kinase B. Using a Cdc25A:ER fusion protein, which can be shuttled from the cytoplasm into the nucleus, the present investigation evidences that the antiapoptotic effect of Cdc25A was restricted to its cytoplasmic localisation in rat 423 cells. In contrast, nuclear Cdc25A overexpression caused dephosphorylation and nuclear retention of the proapoptotic transcription factor Forkhead in rhabdomyosarcoma-like 1 (FKHRL1) in human N.1 ovarian carcinoma cells. This resulted in the increased constitutive expression of the FKHRL1 targets Fas ligand and Bim, and promoted apoptosis. Thus, the Cdc25A oncogene, which was found to be frequently overexpressed in certain human cancers, can increase or decrease the susceptibility to apoptosis depending on the cell-type-specific subcellular distribution.

[The leukocyte migration inhibition test (LMIT) in recurrent herpes simplex labialis. Comparison of the results of treatment with BCG and Levamisole (author's transl)]. / Der Leukocytenmigrationshemmtest (LMIT) bei Herpes simplex labialis recidivans. Vergleich der Therapieerfolge mit BCG und Levamisole.

Forty six patients suffering from recurrent herpes simplex labialis (HSLR) have been treated by either BCG or Levamisole. Therapy was equally successful in both groups: 10 patients remained free of symptoms during the observation period, 11 patients improved clearly and 2 patients did not respond to the therapy. Sensitization of lymphocytes toward herpes simplex virus antigen (HVS-AG) was determined in vitro by means of the leukocyte migration inhibition test (LMIT); test were performed before therapy and in certain intervals after the end of it. While lymphocytes of 10 control persons could be stimulated by HSV-AG, those of patients suffering from HSLR did not react to this antigen before onset of any therapy. Clinical success of therapy correlated well with the in vitro results of the LMIT: after therapy, the lymphocytes of symptom-free and improved patients also reacted in vitro to the antigen added.

Telomeres, telomerase, and myc. An update.

Normal human somatic cells have a finite life span in vivo as well as in vitro and retire into senescence after a predictable time. Cellular senescence is triggered by the activation of two interdependent mechanisms. One induces irreversible cell cycle exit involving activation of two tumorsuppressor genes, p53 and pRb, and the proper time point is indicated by a critical shortening of chromosomal ends due to the end-replication problem of DNA synthesis. The development of a malignant cancer cell is only possible when both mechanisms are circumvented. The majority of human cancers and tumor cell lines produce telomerase, a ribonucleoprotein with two components required for core enzyme activity: telomerase RNA (TR) and a telomerase reverse transcriptase protein (TERT). Telomerase adds hexameric DNA repeats (TTAGGG) to telomeric ends and thus compensates the progressive loss of telomeric sequences inherent to DNA replication. While TR of telomerase is present in almost all human cells, human TERT (hTERT) was found rate limiting for telomerase activity. Ectopic expression of hTERT in otherwise mortal human cells induced efficient elongation of telomeres and permanent cell growth. While hTERT-mediated immortalization seems to have no effect on growth potential and cell cycle check points, it bestows an increased susceptibility to experimental transformation. One oncogene that might activate TERT in the natural context is c-myc. Myc genes are frequently deregulated in human tumors and myc overexpression may cause telomerase reactivation and telomere stabilization which, in turn, would allow permanent proliferation. Is this a general strategy of incipient cancer cells to escape senescence? Several recent observations indicate that other scenarios may be conceived as well.

Demonstration of tumor-associated antigens on tumor cell hybrids by means of the leucocyte-migration-inhibition-test.

The lymphocytes of patients with inoperable bronchus carcinoma were investigated for their in vitro reactivity toward tumor-associated antigens. The leucocyte-migration-inhibition-test (LMIT) was applied using formalinised tumor cells and tumor cell hybrid cell lines. 34 patients with inoperable suqamous cell carcinoma of the lung and 21 healthy controls were tested for their leucocyte sensitisation to these cells. 55 percent of the patients with lung cancer showed a specific LMI toward the E-14 cell line, (an established cell line from a human squamous cell carcinoma of the lung), compared to only 9 percent of the controls. There was about the same percentage of specific reactions of the lymphocytes to three different hybrid cell lines which have only a few human chromosomes left over. There was no reactivity to the parental hamster fibroblasts. It is assumed that the hybrid cells carry the same tumor-associated antigens on their cell surfaces as the E-14 tumor cells.

Effect of tissue culture variables on sister chromatid exchange in a nontransformed rat cell line.

The frequency of sister chromatid exchange (SCE) was determined in a nontransformed diploid rat cell line, FR3T3 , under several tissue culture variables such as cultivation temperature, growth conditions of cells, and concentrations of 5-bromo-2'-deoxyuridine (BrdU). The conclusions to be drawn from these experiments are: (a) The cell growth and mechanisms(s) of SCE formation in FR3T3 cells are largely temperature independent (or efficiently regulated) in the range between 33 and 40.5 degrees C. (b) The concentration limits for BrdU incorporation are 5 to 100 microM; baseline frequency is about 11 SCE/metaphase (constant up to 20 microM BrdU) and increases only moderately at higher BrdU concentrations. (c) Toxic levels of BrdU (150 microM) cause a decrease of SCE rates below that found at 100 microM, presumably due to selective cell death. (d) Keeping cells growth arrested over a long period causes substantial SCE induction after replating. (e) Induced increase of SCEs probably occurs in this manner during the first cell cycle after release from growth arrest. It is no longer detectable after the fourth consecutive cell division.

Chronic application of a tumor promoter to confluent nontransformed rat cells induces the irreversible expression of transformed phenotype.

The hypothesis was put foreward that 'immortalisation' of an established nontransformed cell line might represent one type of 'initiation' in the process of malignant transformation. Therefore, a nontransformed rat fibroblast cell line, FR3T3, was investigated as to whether or not it could be irreversibly transformed by exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA was applied in different treatment schedules at varying growth phases of the cells (logarithmic or stationary growth). The cells' acquisition of transformation-associated properties was studied by means of various transformation assays (saturation densities and formation of dense foci both at low and high serum concentrations, as well as growth in agarose and production of plasminogen activator, PA). We found that a phase of stationary growth plus TPA treatment was decisive for the induction of transformed cell clones. Growth in 0.5% serum was used as the selection procedure. Several isolated foci manifested, in vitro, highly transformed phenotypes. However, only 1 of 12 clones produced PA. All cell lines derived from these clones, proved to be tumorigenic in syngeneic animals. Since the nontransformed, but 'immortal' rat cell line is susceptible (at certain growth conditions) to the tumor promoting activity of TPA, we conclude that induction of 'immortalisation' may, from an operational point of view, correspond to 'initiation', at least in this particular cell line.

The transformation of a rat cell line induced by a tumor promoter occurs without affecting sister chromatid exchange.

Malignant transformation of cells of an established rat cell line, FR3T3, can be achieved by the exclusive treatment of the cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) over a stationary growth period. In the course of the transformation process, sister chromatid exchange (SCE) rates of treated cells were examined at various key-points and compared to control cells. It was found that neither a single TPA treatment nor repeated TPA application during 16-22 consecutive cell-doubling times (which induces substantial alterations of the phenotypes) increased SCE rates in the rat cells. Furthermore, transformed cell clones, which were selected from low-serum cultures and which were found to be tumorigenic in vivo, had SCE rates in the same range as the parental nontransformed FR3T3 cells. It is, therefore, concluded that malignant transformation of the rat cells - induced by TPA - occurs without affecting such large-scale DNA rearrangements as are detectable by means of the SCE method.

The detection and measurement of oncodevelopmental proteins in gynecological malignant disease.

Data from the literature and our own measurements with alpha-fetoprotein (AFP), carcinoembryonal antigen (CEA), alpha 2-pregnancy-associated globulin (alpha 2-PAG), human chorionic gonadotropin (HCG), human placental lactogen (HPL) and pregnancy-specific beta 1-glycoprotein (SP-1) are related to clinical data. Only AFP in endodermal tumors and HCG in germ cell carcinomas can be used for clinical monitoring.

Increase of sister chromatid exchange formation induced by diethylstilbestrol.

The latent appearance of genital tract tumors in young women is highly correlated with prenatal exposure to synthetic estrogens such as diethylstilbestrol (DES). To extend the information on the mutagenic effect of DES, the frequency of sister chromatid exchange (SCE) was determined in bone marrow cells of Sprague-Dawley rats and in cultured FR3T3 rat fibroblasts. In this study a significant increase of SCE frequency after the application of different concentrations of DES could be observed both in vivo and in vitro.

Sister chromatid exchange in FR3T3 rat fibroblasts transformed by Simian virus 40.

The frequency of Sister Chromatid Exchange (SCE) was determined at low (33 degrees C) and high (40.5 degrees C) temperatures in cell lines derived from FR3T3 rat fibroblast cells after transformation either with Wild-Type Simian Virus 40 (SV40-WT), with an origin-defective SV40 (SV40-ori-), or with the early temperature-sensitive mutant tsA30. Of these cell lines, SV40-WT-, SV40-ori--, and one class of tsA30-transformants (A-type) express the transformed phenotype both at 33 and 40.5 degrees C. The other tsA30-transformants (N-type) revert to a normal phenotype at high temperature. As compared with normal FR3T3 cells, all transformants exhibited, at 33 degrees C, increased numbers of metaphases with high SCE rates. At 40.5 degrees C, all cell lines which expressed a transformed phenotype (SV40-WT, tsA30 type A, SV40-ori-) exhibited substantially increased SCE rates. That this increase was not related to a possible induction of viral replication by BrdU, was proven by Southern blot analysis and by SCE data on SV40-ori--transformed cells. By contrast, no such temperature-induced increase of SCE rates was observed in tsA30-transformants of type N.