Antitumor activity was measured by a microtetrazolium assay following a 5-day exposure to vitamin C (VC), vitamin K₃ (VK₃), or a vitamin combination with a VC:VK₃ ratio of 100:1. Values are the mean ± standard error of the mean of three experiments, with six readings per experiment. Fractional inhibitory concentration index = CD₅₀^{A comb}/CD₅₀^{A alone} + CD₅₀^{B comb}/CD₅₀^{B alone}, where CD₅₀^{A alone} and CD₅₀^{B alone} are 50% cytopathic doses of each vitamin alone; CD₅₀^{A comb} and CD₅₀^{B comb} are the 50% cytotoxic doses of the vitamins administered together.

Ultrastructural topography of the nucleoli of autoschizic bladder carcinoma cells that have been treated for 4 hr with PBS (A), VC (B), VK₃ (C), or Apatone (the vitamin combination) (D). (A) Sham-treated bladder cancer cell possesses a voluminous, reticulated nucleolus with a dispersed dense fibrillar component (D) and many interstices (I). Several small fibrillar centers can be seen among the granular component (G), which is dispersed throughout the nucleus. The chromatin (C) is associated with the nuclear membrane. (B) VC–treated cells display a more contrasted aspect with a prominent granular component (G), but the nucleolar interstices (I), dense fibrillar (D), and the fibrillar centers (F) are still detectable. The nucleolus is still in contact with the chromatin (C) associated with the nuclear envelope. (C) VK₃–treated cells show nucleoli with a more compact aspect. The fibrillar centers (F) appear less numerous in control cells, and a large proportion of the nucleolar mass is the granular component (G). Nucleolar interstices (I) and dense fibrillar components (D) are visible. The overall nucleolar structure is more electron densely contrasted than the control and is no longer in contact with the nuclear envelope. (D) Apatone-treated cell shows a nucleolus as a compact, ovoid body consisting primarily of a granular component (G). A limited number of interstices (I) are visible. The nucleolar-associated, condensed chromatin (C) appears to have been extruded from the body of the nucleolus and is associated with the outside of the nucleolus. Bar = 0.5 μm.

A series of photomicrographs made after 1-, 2-, 3-, or 4-hr Apatone treatment. This figure illustrates the progressive segregation of the nucleolar components, where the electron densely contrasted chromatin (C) (DNA labeled) associates and interacts with the other nucleolar components, along with chromatin associated with the inner nuclear membrane (B,C). The nucleolonema remains somewhat as a main mass of granular component (G), unlabeled by DNA antibody, first with small interstices (I), and finally forming a compact aggregate (D). The dense component is discrete and is detected in B only. From a series of chromatin fingers interacting and penetrating the nucleolus, as shown in A,B, the granular component becomes more and more compacted, whereas the chromatin component appears to be partially to totally extruded and to exit the granular component (D). Bar = 5 μm.

T24 bladder carcinoma cells were treated for 4 hr with PBS (A,C,E,G) or with Apatone (B,D,F,H). The localization of the nucleolar proteins nucleophosmin/B23 (A,B), RH-II/Gu (C,D), p120 (E,F), and fibrillarin (G,H) were determined by indirect immunofluorescent labeling. After Apatone treatment, nucleolar compaction was observed. A small amount of redistribution was observed with RH-II/Gu. Originally, the overall distribution of the fibrillarin matched with the fibrillar centers. Following Apatone treatment, there was a redistribution of fibrillarin from the nucleolar fibrillar centers and adjacent regions to the whole nucleolar body. Neither nucleophosmin/B23 nor p120 exhibited any redistribution.

T24 bladder carcinoma cells were treated with PBS (top panels) or PBS containing 1000 U/ml of DNase I (bottom panels). The nuclei were stained with 4′,6-diamidino-2-phenylindole (left panels), or the localization of fibrillarin was determined by indirect immunofluorescent labeling (right panels). DNase I treatment produced a relocalization of fibrillarin that closely resembled that induced by Apatone treatment.

T24 cells were sham treated (A) or exposed to the vitamin combination at its 90% cytotoxic doses (B) for 1 hr. The vitamins were then removed, and the cells were washed twice with PBS and overlain with culture medium. After 24 hr, the cells were harvested, and DNA ploidy and cell cycle analysis was performed on an Ortho Cytoron flow cytometer. The data from 2 × 10⁴ cells were collected (when possible), stored, and analyzed using ModFit Cell Cycle Analysis. Sham-treated T24 cells served as the negative control. Human foreskin fibroblasts served as diploid control. (A) The flow cytometry trace of a mixture of diploid human foreskin fibroblasts (HSF) and bladder cancer cells (T24) which allows determination of the ploidy of the T24 cells with respect to normal diploid cells as well as the cell cycle distribution of both cell types following sham treatment. (B) The flow cytometry trace of T24 following Apatone treatment shows the enrichment in both the G₀-G₁ and the G₂-M populations as well as the appearance of an aneuploid peak indicative of autoschizis.

Distribution of DNA immunogold (diameter = 10 nm) within the nucleoli of T24 bladder carcinoma cells treated for 4 hr with PBS (sham), menadione, ascorbate, or Apatone. (A) Example of a sham-treated T24 cell showing a large reticulated nucleolus with a dispersed dense fibrillar component (D) and several fibrillar centers (FC) scattered throughout the granular region (G). The chromatin (C) is characteristically labeled, and DNA can be detected throughout the nucleolus and is detected across the nucleolus, forming a large network from one side to the opposite side of the nuclear envelope. (B) Following menadione treatment, the nucleolus acquires a more compact morphology, and the dense fibrillar component (D) forms a large, condensed arched structure surrounded by the granular component (G). The outermost electron-dense component is the chromatin (C) and is properly labeled. Both fibrillar centers show a few labels adjacent to the dense component. (C) Ascorbate-treated T24 nucleolus shows DNA labels in packets in narrow interstices (I) of the reticulated and compact mass made by the granular region (G) and throughout the nucleoplasm. The small arrows denote thread-like dense fibrillar component. (D) T24 cells treated by Apatone demonstrate the most compact nucleolus with large interstices (I) among the granular component (G). Only a few labeled strands of chromatin appear centrifugal to the compact nucleolar mass. No labels are found in the granular component. Rare DNA labels can be found in the nucleoplasm. Bar = 5 μm.

Distribution of RNA immunogold (diameter = 5 nm) within the nucleoli of T24 bladder carcinoma cells treated for 4 hr with PBS (sham), menadione, ascorbate, or Apatone. (A) Control cells show a granular region (G) of a nucleolus completely labeled in an even distribution of isolated or aggregated immunogold particles. A general fine dispersion of labels can be found around the nucleolar area and throughout the nucleoplasm. (B) Following a 4-hr menadione treatment, the anti-RNA immunolabelings are distributed over the granular component (G) of the nucleolus, and some labels can be found at the edges of the nucleolus and throughout the nucleoplasm. Large interstices (I) can be found. The dense, thick component is unlabeled. (C) After 4 hr of ascorbate treatment, the distribution of labels is in packets on the granular reticulated and compacted granular (G) region, at the edge of interstices (I) and rare dense component (D) material of the nucleolus, and some labels are dispersed throughout the nucleoplasm. (D) After 4 hr of combined treatment by Apatone, the segregation of the nucleolar components is visible. Although the entire granular component (G) appears in the form of compacted fine paracrystalline lines of ∼7–10 nm in thickness, and is labeled, mainly at the edges of the tightly closing interstices (I), no fibrillar center region can be found, and a dispersed labeling is found outside the nucleolar mass. Bar = 5 μm.

T24 bladder carcinoma cells were treated for 4 hr with PBS (A), VC (B), VK₃ (C), or the Apatone combination (D). The localization of fibrillarin was determined by indirect immunofluorescent labeling. In cells treated with either VC or VK₃, some relocalization of fibrillarin was observed. However, in cells treated with both VC and VK₃, a marked relocalization of fibrillarin was observed.