Inaccessibility of the Euplotes telomere binding protein.

The telomere binding protein (TP) from the macronucleus of the ciliate Euplotes eurystomus was purified by removal of tenaciously bound DNA with hydroxylapatite, and the purified TP partially sequenced. Rabbit antiserum was generated against a synthetic peptide of 14 amino acids at the amino-terminus of the TP. This antiserum was employed to examine the accessibility of TP antigenic determinants in nuclei and chromatin. Immunofluorescent staining of isolated macronuclei revealed only weak reactivity with specific antiserum. Reactivity within replication bands was demonstrated, and could be augmented by preparation of nuclear scaffolds. Employing a dot immunoblot analysis, the amino-terminal antigenic determinants of TP were revealed after extraction of histone H1 (and some nonhistones). A different aspect of TP inaccessibility was demonstrated by immunoblot analysis of trypsin-treated macronuclei and chromatin; TP was considerably less susceptible to digestion by trypsin than were histones H1 and H3. The relative inaccessibility of TP was not a consequence of chromatin higher-order structure, since soluble macronuclear chromatin in low salt exhibited the same burying of antigenic determinants by dot blot analysis, and the same decreased susceptibility to trypsin, as did isolated nuclei. Electron microscopy of soluble macronuclear chromatin spread in low salt revealed that most telomeres appear unfolded, without stable higher-order structure. The mechanisms for the relative inaccessibility of TP are not yet known, but probably arise as a consequence of the strong interactions of TP with the telomere nucleotide sequence and additional interactions of TP with various chromatin proteins, perhaps including histone H1.

Proliferating cell nuclear antigen/cyclin in the ciliate Euplotes eurystomus: localization in the replication band and in micronuclei.

Human autoimmune sera specific for proliferating cell nuclear antigen (PCNA)/cyclin (auxiliary protein for DNA polymerase delta) demonstrated the presence of epitopes within the macro- and micronuclei of the hypotrichous ciliated protozoa Euplotes eurystomus. Tightly bound PCNA/cyclin was localized at the site of DNA synthesis in macronuclei, the rear zone of the replication band. Starvation or heat shock, conditions that reduce macronuclear replication, resulted in a decrease of PCNA/cyclin in replication bands. Micronuclei also exhibited PCNA/cyclin localization which persisted for a large proportion of the vegetative cell cycle and exhibited significant resistance to adverse culture conditions. Immunoprecipitation of 35S-labeled soluble Euplotes proteins with PCNA/cyclin autoimmune sera revealed a spectrum of low molecular mass proteins. PCNA/cyclin-like proteins have now been observed in the widely divergent species: human, rat, amphibian, yeast, and ciliated protozoa.

An H1-like protein from the macronucleus of Euplotes eurystomus.

An H1-like protein has been purified from the macronucleus (MAC) of the hypotrichous ciliated protozoan, Euplotes eurystomus. It is present in amounts comparable to the inner histones and is extracted by treatment with 5% perchloric acid or 0.65 M NaCl, but not by 0.35 M NaCl. Treatment of soluble MAC chromatin with the ionic exchange resin AG 50W-X2 in 80 mM NaCl removes MAC H1 and yields H1-depleted chromatin. Mac H1 is lysine-rich and deficient in acidic amino acids. The stoichiometry of the H1 protein is reduced in mononucleosome preparations, consistent with its postulated interaction with linker DNA regions. Thermal denaturation and circular dichroism studies reveal that H1-depleted chromatin contains a larger portion of destabilized DNA than control chromatin. The molecular weight of Euplotes MAC H1 is significantly smaller than most reported H1 proteins. Comparisons are made with extracts of macronuclei from other hypotrichous ciliated protozoa and published reports of other lower eukaryotes.

Temporal advancement of diethylnitrosamine carcinogenesis in aging mice.

Female BALB/c mice were given diethylnitrosamine (DEN) in their drinking water beginning at 2.5, 9.5, and 17 months of age (cumulative dose approximately 300 to 400 mg/kg body weight) or were untreated. Median times of death for the treatment groups were 193, 168, and 125 days, respectively, after cessation of DEN treatment and were significantly different (p less than .01). Induced tumors in the three respective age groups were of squamous forestomach (88, 87, and 84%), vascular tumors of the liver (11, 13, and 16%), and adenomas of the lung (65, 56, and 54%). Controls had no forestomach or liver tumors and relatively low incidences of lung tumors. The fact that aging mice have similar incidences and types of tumors of the same size and in the same tissues, but at an earlier time, shows that (1) DEN is carcinogenic in aging BALB/c mice; (2) age at treatment does not alter the tumor-susceptible tissue nor types of tumors after DEN treatment; (3) tumor incidences are not affected by age at time of treatment; (4) mice die earlier with induced tumors with increasing age at time of treatment; (5) age-matched non-DEN-treated mice die from different diseases (leukemias) than do DEN-treated mice (stomach and liver tumors). These observations may be related, in part, to an identified age-dependent decrease in immunocompetency or to other age-related changes, such as vascular or hormonal, which could explain temporal advancement in the tumorigenic process.

Immunodepression, a noncontributor to radiation-induced leukemogenesis of the RFM mouse.

Following a single acute exposure to 300 R of X-rays at 6 weeks old, approximately 65% of female RFM mice die of thymic lymphoma during the first year of life. In contrast, nonirradiated animals do not die of this neoplasm during this same period. To determine if immediate immunological restoration is of significance in interrupting the inductive process, we injected 50 X 10(6) syngeneic spleen or bone marrow cells into these animals immediately following X-irradiation. Restoration of immunocompetence as measured by both humoral and cell-mediated parameters was more rapid in spleen cell-reconstituted animals than in bone marrow-treated animals; however, spleen cells failed to protect the mice against the irradiation-induced thymic lymphomas. In contrast, although there was no significant difference in the immunological recovery of bone marrow-reconstituted animals and animals that received only irradiation, mortality was dramatically reduced as a result of marrow injection. Therefore, although immunodepression is an inherent component, it cannot be considered as a critical obligatory requirement in the pathogenesis of radiation-induced thymic lymphomas of RFM mice.

A positive correlation between declining immune competence and early mortality associated with diethylnitrosamine carcinogenesis in aging mice.

The effect of age (2.5, 9.5, and 17 months) at time of treatment upon diethylnitrosamine (DEN) carcinogenesis and immune competence has been assessed in female BALB/c mice. Median times of death were 193, 168, and 125 days, respectively, after termination of DEN treatment. Immune competence as a measure by both cell-mediated and humoral immune parameters immediately after DEN treatment was not significantly different among treated and age-matched non-treated control animals. In contrast, a significant age-related decline in immune competence was seen in both DEN-treated and non-treated controls, thereby demonstrating a direct and positive correlation between the natural age-related decrease in immune competence and cancer-induced advanced mortality.

A multiple-parameter comparison of immunocompetence and tumor resistance in aged BALB/c mice.

Several parameters of cell-mediated and humoral immunity were measured in young-adult and aged BALB/c mice and compared with resistance to the ascites form of intraperitoneal induced P815 mastocytoma. It was found that the age-related decline of in vitro phytohemagglutinin (PHA) responsiveness by spleen cells approximated the marked decrease of old mice to tumor-cell challenge and approached that characteristically seen in humoral immunity. Thus, decreased PHA responsiveness of splenic lymphocytes provided as sensitive an estimate of the age-related decline of immunocompetence in old mice as other classical parameters of cell-mediated immunity (e.g. graft-vs-host reaction or in vivo cellular proliferation of parental spleen cells in lethally-irradiated F1 recipients). Results could be interpreted to represent a decreased ability of noncycling T-cells to be released to a functional cycling state.

The late effects of selected immunosuppressants on immunocompetence, disease incidence, and mean life-span. I. Humoral immune activity.

The effect of different immunosuppressive treatments during young adulthood or humoral immune competence late in life was determined. It was found that the marked reduction in humoral immune competence in aged mice is further compromised when severe insults are administered early in life. Thus, thymectomy, splenectomy, and sublethal X-irradiation produced lasting immunodepression as measured (1) in situ and (2) by the hemolysin, direct and indirect plaque forming cell responses of adoptively transferred spleen cells. In contrast, treatment with cyclophosphamide and cortisone acetate were without effect, indicating that drug-damaged cells of the immune system were replaced by competent cells during the course of time. Decrease in immune competence of aged thymectomized animals could not be correlated with a decrease in numbers of theta-bearing T or immunoglobulin receptor-bearing B lymphocytes. The significance of the observed unequal effects of these immunosuppressants on immune competence, as they relate to disease incidence and life expectancy, are dealt with in the third paper in this series.