Patricia L. Witt

Structure of the mammalian kinetochore

The structure of the mammalian trilaminar kinetocnore was investigated using stereo electron microscopy of chromosomes in hypotonie solutions which unraveled the chromosome but maintained microtubules. Mouse and Chinese hamster ovary cells were arrested in Colcemid and allowed to reform microtubules after Colcemid was removed. Recovered cells were then swelled, lysed or spread in hypotonic solutions which contained D2O to preserve microtubules. The chromosomes were observed in thin and thick sections and as whole mounts using high voltage electron microscopy. Bundles of microtubules were seen directly attached to chromatin, indicating that the kinetochore outer layer represents a differential arrangement of chromatin, continuous with the body of the chromosome. In cells fixed without pretreatment, the outer layer could be seen to be composed of hairpin loops of chromatin stacked together to form a solid layer. The hypotonically-induced unraveling of the outer layer was found to be reversible, and the typical 300 nm thick disk reformed when cells were returned to isotonic solutions. Short microtubules, newly nucleated after Colcemid removal, were found not to be attached to the kinetochore outer layer, but were situated in the fibrous corona on the external surface of the outer layer. This was verified by observations of thick sections in stereo which made it possible to identify microtubule ends within the section. Thus, kinetochore microtubules are nucleated within the fibrous corona, and subsequently become attached to the outer layer.

Origin of kinetochore microtubules in Chinese hamster ovary cells

We have attempted to determine whether chromosomal microtubules arise by kinetochore nucleation or by attachment of pre-existing microtubules. The appearance of new microtubules was investigated in vivo on kinetochores to which microtubules had not previously been attached. The mitotic apparatus of Chinese hamster ovary cells was reconstructed in three dimensions from 0.25 μm thick serial sections, and the location of chromosomes, kinetochore outer disks, centrioles, virus-like particles and microtubules determined. Central to the interpretation of these data is a synchronization scheme in which cells entered Colcemid arrest without forming mitotic microtubules. Cells were synchronized by the excess thymidine method and exposed to 0.3 μg/ml Colcemid for 8 h. Electron microscopic examination showed that this Colcemid concentration eliminated all microtubules. Mitotic cells were collected by shaking off, and cell counts showed that over 95% of the cells were in interphase when treatment began and thus were arrested without the kinetochores having been previously attached to microtubules. Cells were then incubated in fresh medium and fixed for high voltage electron microscopy at intervals during recovery. — In early stages of recovery, short microtubules were observed near and in contact with kinetochores and surrounding centrioles. Microtubules were associated with kinetochores facing away from centrosomes and far from any centrosomal microtubules, and thus were not of centrosomal origin. At a later stage of recovery, long parallel bundles of microtubules, terminating in the kinetochore outer disk, extended from kinetochores both toward and away from centrosomes. Because microtubules had never been attached to kinetochores, the possibility that kinetochore microtubles were initiated by microtubule stubs resistant to Colcemid was eliminated. Therefore we conclude that mammalian kinetochores can initiate microtubules in vivo, thus serving as microtubule organizing centers for the mitotic spindle, and that formation of kinetochore-microtubule bundles is not dependent on centrosomal activity.

Structure of kinetochore fibers: Microtubule continuity and inter-microtubule bridges

To understand how microtubules interact in forming the mitotic apparatus and orienting and moving chromosomes, the precise arrangement of microtubules in kinetochore fibers in Chinese hamster ovary cells was examined. Individual microtubules were traced, using high voltage electron microscopy of serial 0.25 μm sections, from the kinetochore toward the pole. Microtubule arrangement in kinetochore fibers in untreated mitotic cells and in cells recovering from Colcemid arrest were similar in two respects: the number of microtubules per kinetochore (mean 14 and 12, respectively) and the nearest neighbor intermicrotubule distance (mean∼90 nm). In Colcemid recovered cells, over 90% of the microtubules in kinetochore fibers were attached to the kinetochore (i.e. kinetochore microtubules) and extended most or all of the distance to the pole. Few free microtubules were present in the kinetochore fibers; most non-kinetochore microtubles terminated in the pole. Since kinetochores in this Colcemid-recovered system have been demonstrated to nucleate microtubules (Witt et al., 1980), it seems likely that most if not all of these kinetochore microtubules originated at the kinetochore. Some of the reconstructed kinetochore fibers were attached to chromosomes with bipolar orientation, suggesting that kinetochore microtubules need not interact with many polar microtubules for orientation to occur. In Colcemid recovered cells lysed to reduce cytoplasmic background, microtubules in kinetochore fibers were preferentially preserved. The parallel and near-hexagonal order typical of microtubules in kinetochore fibers was maintained, as was the number of kinetochore microtubules (mean, 13). The intermicrotubule distance was slightly reduced in lysed cells (mean, 60 nm). Crossbridges about 5 nm wide and 30–40 nm long were visible in kinetochore fibers of lysed cells. Such crossbridges probably contribute to the stabilization and parallel order of microtubules in kinetochore fibers, and may have a functional role as well.

A direct comparison of biological response modulation and clinical side effects by interferon-beta ser, interferon-gamma, or the combination of interferons beta ser and gamma in humans.

To directly compare clinical side effects and biological response modification, IFN-beta ser, IFN-gamma, or the combination of IFN-beta ser plus IFN-gamma was administered to 21 cancer patients. Each IFN or the combination was given intravenously on days 1, 8, and 15 in varied order. Each IFN and the combination resulted in significant (P less than 0.05) modulation of IFN-induced proteins. IFN-beta ser was more effective than IFN-gamma in enhancing 2-5A synthetase activity (P = 0.001). IFN-gamma was more effective than IFN-beta ser in enhancing serum beta 2 microglobulin expression (P = 0.05) and indoleamine dioxygenase activity, as assessed by decreased serum tryptophan (P = 0.03). The combination enhanced tryptophan catabolism more effectively than IFN-beta ser in a dose-dependent manner (P less than 0.03). IFN-beta ser/IFN-gamma did not potentiate natural killer cells or antibody-dependent cellular toxicity (ADCC). IFN-beta ser/IFN-gamma enhanced monocyte guanylate cyclase activity, as assessed by serum neopterin, more effectively than IFN-gamma alone (P = 0.005). Both IFNs and the combination resulted in increases in HLA class II expression on monocytes. However, no significant difference in the level of induction of HLA DQ and HLA DR expression between IFN-beta ser/IFN-gamma and either IFN-beta ser or IFN-gamma was noted. Although frequency and servity of side effects of IFN-beta ser, IFN-gamma, or the combination were dose related, induction of induced proteins (with exception of influences on tryptophan catabolism) were not a function of dose administered over the 10-fold range. Continued treatment with the combination intravenously three times a week for 4 wk sustained but did not further potentiate, most of the changes in interferon-induced proteins. Thus, IFN-beta ser and IFN-gamma each resulted in effective and essentially equivalent patterns of induction of induced proteins. When combined, however, these IFNs did not result in potentiation of biological response modification in vivo.

Clinical and biologic effects of combination therapy with gamma‐interferon and tumor necrosis factor

Tumor necrosis factor (TNF) and gamma‐interferon (γ‐IFN) are cytokines with synergistic biologic and antiproliferative effects in vitro and in mouse models. The biologic effects of the combination of TNF and γ‐IFN, however, have not been studied well in humans. A Phase I trial was conducted of TNF and γ‐IFN therapy in 24 patients with advanced malignancies to determine the tolerability of the combination and the biologic effects of TNF and γ‐IFN in vivo. Both TNF and γ‐IFN were administered as 30‐minute intravenous infusions three times per week. Doses of TNF ranged from 25 to 100 μg/m2; all patients received 100 μg/m2 of γ‐IFN. Dose‐limiting toxicity consisted primarily of orthostatic hypotension and constitutional symptoms. The maximum tolerated dose level (MTDL) of 50 μg/m2 of TNF and 100 μg/m2 of IFN‐γ was less than the maximum tolerated dose (MTD) observed in previous Phase I trials of γ‐IFN and TNF alone. Biologic responses were studied in seven patients treated at the MTDL. Serum interleukin‐2 receptor levels and neopterin secretion were enhanced significantly 24 hours after therapy (P = 0.002); enhancement of monocyte Fc receptor levels had borderline statistical significance (P = 0.07). With the exception of the mean fluorescent intensity on monocytes positive for histocompatibility antigen HLA‐DR (P = 0.03), HLA Class I and II cell surface protein expression was not increased. The combination significantly enhanced indoleamine dioxygenase activity and serum β2‐microglobulin expression (P < 0.04) but not 2′,5′‐oligoadenylate synthetase activity, bactericidal function, or chemiluminescence. These results were compared retrospectively with those observed in previous Phase I trials of γ‐IFN and TNF alone. The combination of TNF and γ‐IFN significantly increased urinary kynurenine levels more than either TNF alone or γ‐IFN alone. Given the limitations inherent in any retrospective analysis, however, the enhancement in the other biologic parameters measured at the MTDL during this trial did not differ significantly from the changes observed at the MTD of either TNF or γ‐IFN alone. It was concluded that the combination of TNF and γ‐IFN, when administered at the MTDL of the combination, does not offer any enhancement in biologic responses over either agent alone.

Inhibition of hormone-dependent and independent breast cancer cell growth in vivo and in vitro with the antiestrogen toremifene and recombinant human interferon-α2

The antiproliferative action of the antiestrogen toremifene and recombinant human interferon-α2a (IFN-α2a) were examined on human breast cancer cell lines grown in culture and in the athymic mouse.Solid tumors grew from an inoculation of a 99:1 ratio of hormone dependent (MCF-7) and hormone independent (MDA-MB-231) breast cancer cells without estrogen administration. However, estradiol supplementation significantly increased the rate of tumor growth. The daily administration of 1.35 × 106 U of recombinant human IFN-α2a resulted in a marked reduction of tumor growth in both estradiol-treated and non-treated mice. Toremifene administration (130 µg/day from a sustained release preparation) markedly inhibited estradiol stimulation of mouse uterine weight and partially reduced estradiol-stimulated tumor growth. The combination of IFN-α2a (1.35 × 106 u/day) with toremifene (130 µg/day) reduced estradiol-stimulated growth much below that of toremifene alone but not below that seen with interferon alone.Toremifene (10−10-10−6M) did not inhibit the growth of hormone-independent MDA-MB-231 breast cancer cellsin vitro whereas it did inhibit the growth of hormone-dependent MCF-7 cells in phenol red containing media. IFN-α2a (1–10,000 u) inhibited the growth of both MCF-7 and MDA-MB-231 cells in culture; however, MCF-7 cells were approximately 10-fold more sensitive to interferon inhibition. This was consistent with the MCF-7 cells showing a greater sensitivity to interferon than MDA-MB-231 cells in the induction of 2′5′-oligoadenylate synthetase.The heterogeneous tumor model in the athymic mouse suggests that differential sensitivities of breast cancer cells to the antiproliferative actions of interferon may influence the effectiveness of combination therapies.

Unequal distribution of DNA in the macronuclear division of the ciliate Euplotes eurystomus

During asexual fission in the ciliate Euplotes eurystomus, the macronucleus divides amitotically. The macronucleus was found to divide unequally, yielding sister pairs having a mean difference in DNA content of 11.6%. DNA content was determined by the Feulgen reaction using a fluorescent Schiffs reagent, and measuring fluorescence by cytophotometry. Variability in macronuclear DNA content was also examined in randomly-paired non-sister cells, and found to be greater than in sister cells. This greater variability could be due to accumulation of differences over a number of divisions, or to interclonal differences in equality of division. Two categories of non-sister cells were examined: recently divided, and “parents” constructed by averaging the DNA contents of progeny. Both showed similar variability in quantity of macronuclear DNA. The fact that cells surviving to divide showed no less variability in amount of DNA than cells immediately after division suggests that extremes in amounts of DNA resulting from unequal division are not selected against.

The October 29th Group: Defining a feminist science

Synopsis A group of scientists and those interested in the sciences, all feminists, has been meeting for six years at the University of Wisconsin-Madison to discuss and define a feminist critique of science. We consider science an important area for feminist investigation for several reasons. Scientific research is funded, conducted, and applied for the goals of those with political power. Patriarchy throughout history has used science to justify itself, for example, assigning sociobiological explanations to class, race, or gender inequalities. Many of the characteristics we value would generally be considered part of “dgood” science. These include sharing of information, availability of funding for projects representing diverse points of view, opportunity regardless of gender, race, religion, and judging work on its merits rather than the status of the authors. Other criticisms are more specifically feminist, examining how patriarchal politics and worldview are played out in science. Some examples include an emphasis on the control of nature rather than understanding, a search for “master” molecules presupposing a hierarchical rather than an interactive model, or reliance on reductionism, quantification, and linear thinking while denegating other modes of obtaining and organizing information. We emphasize ways to bring a feminist commitment and philosophy into the laboratory and outline one possible mechanism for transformation of science to more egalitarian principles. Our group represents for us an example of feminist science in action, in its emphasis on open discussion, support as well as criticism, and awareness of the relationship between means and ends, theory and practice.