Aneuploid Nuclei Research Articles

Evaluation of genomic changes in a large series of malignant ovarian germ cell tumors—relation to clinicopathologic variables

Malignant ovarian germ cell tumors (mOGCT) affect women in their reproductive years, making fertility-saving treatment important. A reliable prediction of the clinical behavior is essential for an optimal therapeutic approach. The genetic changes and molecular mechanisms underlying these rare tumors remain poorly understood. To address these issues, we performed DNA ploidy analysis by high-resolution image cytometry in a series of 47 mOGCT and correlated the findings with the DNA copy number changes detected by comparative genomic hybridization (CGH) and clinical outcome. Of 47 tumors, 15 were diploid, 14 were tetraploid, 2 were polyploid, and 13 were aneuploid. All the immature teratomas were diploid, in contrast to the dysgerminomas and endodermal sinus tumors. The International Federation of Gynecology and Obstetrics (FIGO) staging, residual tumors after surgery, and DNA ploidy distribution were significant, independent prognostic factors in survival analysis. The study revealed that the number of DNA copy number aberrations was increased in tetraploid and aneuploid tumors as compared to diploid tumors. Furthermore, a high percentage of aneuploid nuclei in a sample were associated with a complex CGH profile of the tumor in question. The present study confirms that DNA aneuploidy assessment by image analysis may be linked to genetic instability, which is detected as genetic aberrations by CGH. DNA ploidy gives significant prognostic information in addition to the clinical stage in mOGCT with FIGO stage II–IV.

Drosophila Checkpoint Kinase 2 Couples Centrosome Function and Spindle Assembly to Genomic Integrity

In syncytial Drosophila embryos, damaged or incompletely replicated DNA triggers centrosome disruption in mitosis, leading to defects in spindle assembly and anaphase chromosome segregation. The damaged nuclei drop from the cortex and are not incorporated into the cells that form the embryo proper. A null mutation in the Drosophila checkpoint kinase 2 tumor suppressor homolog (DmChk2) blocks this mitotic response to DNA lesions and also prevents loss of defective nuclei from the cortex. In addition, DNA damage leads to increased DmChk2 localization to the centrosome and spindle microtubules. DmChk2 is therefore essential for a “mitotic catastrophe” signal that disrupts centrosome function in response to genotoxic stress and ensures that mutant and aneuploid nuclei are eliminated from the embryonic precursor pool.

Combined DNA flow cytometry and sorting with k-ras2 mutation spectrum analysis and the prognosis of human sporadic colorectal cancer.

Activation of the k-ras2 pathways and chromosomal instability leading to aneuploidy in human sporadic colorectal cancer (sCRC) is essential to the tumor cell ability to survive, grow, and metastatize. The study included 135 patients with sCRC who were followed up for a median of 72 months. Multiple fresh-frozen fragments obtained from superficial and invasive areas of the tumors were mixed and used to detect the degree of DNA aneuploidy (DNA index [DI]) and S-phase fraction by two scatter signals and 4,6-diamidino-2-phenylindole-2-hydrocloride (DAPI) fluorescence flow cytometry (FCM). PCR amplification and k-ras2 mutation spectrum analysis were performed using enriched epithelial nuclei after sorting DNA aneuploid nuclei and DNA diploid nuclei from which tissue-infiltrating lymphocytes were absent. DNA aneuploidy was detected in 98 (73%) and k-ras2 mutations in 54 cases (40%). Univariate analyses of overall survival with both Dukes' A to D or B to C series of cases showed that DNA multiple aneuploidy, k-ras2 mutations, older age, and distal site, but not increased S-phase fraction, were predictive of worse outcome. Multivariate Cox models strongly indicated that k-ras2 mutations, but neither single nor multiple DNA aneuploidy, were an independent prognostic factor in both series of patients. In particular, with B and C Dukes' stage patients (n = 110), the relative risk (RR) of death was above 2.5 with k-ras2 mutations and above 3 with the G-->C/T transversions. Combined FCM and k-ras2 analysis may be used to predict long-term increased risk of death in sCRC patients.

Monochloramine inhibits etoposide-induced apoptosis with an increase in DNA aberration

Monochloramine (NH 2Cl) is a physiological oxidant produced by activated neutrophils, and it affects apoptosis signaling. We studied the effects of NH 2Cl on the cell death induced by etoposide, a widely used anticancer agent that is directed to DNA topoisomerase II. Jurkat T cells, a human acute T cell leukemia cell line, were pretreated with 70 μM of NH 2Cl for 10 min. After 24 h, 5–30 μM of etoposide was added to the NH 2Cl pretreated and control cells, and their apoptosis, caspase activity, cell morphology, and cellular DNA contents were measured. NH 2Cl pretreatment significantly inhibited apoptosis and caspase activation induced by etoposide or camptothecin, a DNA topoisomerase I poison, but not by staurosporine or Fas stimulation. The apoptosis inhibition actually resulted in the proliferation of the survived cells and, notably, the survived cells showed more aberrant morphology, such as variation in nuclear size, nuclear fragments, and multinucleated cells. DNA content analysis of the survived cells showed an increase in aneuploid nuclei. Cell cycle analysis after 24 h of NH 2Cl treatment showed a significant decrease in S phase cells with a concurrent increase in G 0/G 1 phase cells, which suggested that NH 2Cl induced G 1 arrest. Using synchronized Jurkat cells, etoposide and camptothecin were found to be particularly cytotoxic to S phase cells, whereas staurosporine and Fas stimulation were not. Thus NH 2Cl-induced G 1 arrest was a likely cause of the observed resistance to etoposide. These observations suggested that inflammation-derived oxidants may make the tumor cells more resistant to etoposide and increase the risk of tumor progression and the development of secondary tumors by increasing the survival of DNA damage-bearing cells.

Paternal Chromosome Incorporation into the Zygote Nucleus Is Controlled by maternal haploid in Drosophila

maternal haploid (mh) is a strict maternal effect mutation that causes the production of haploid gynogenetic embryos (eggs are fertilized but only maternal chromosomes participate in development). We conducted a cytological analysis of fertilization and early development in mh eggs to elucidate the mechanism of paternal chromosome elimination. In mh eggs, as in wild-type eggs, male and female pronuclei migrate and appose, the first mitotic spindle forms, and both parental sets of chromosomes congress on the metaphase plate. In contrast to control eggs, mh paternal sister chromatids fail to separate in anaphase of the first division. As a consequence the paternal chromatin stretches and forms a bridge in telophase. During the first three embryonic divisions, damaged paternal chromosomes are progressively eliminated from the spindles that organize around maternal chromosomes. A majority of mh embryos do not survive the deleterious presence of aneuploid nuclei and rapidly arrest their development. The rest of mh embryos develop as haploid gynogenetic embryos and die before hatching. The mh phenotype is highly reminiscent of the early developmental defects observed in eggs fertilized by ms(3)K81 mutant males and in eggs produced in incompatible crosses of Drosophila harboring the endosymbiont bacteria Wolbachia.

Germ-Cell Nondisjunction in Testes Biopsies of Men With Idiopathic Infertility

Intracytoplasmic sperm injection (ICSI) has been used in combination with testicular sperm extraction to achieve pregnancies in couples with severe male-factor infertility, yet many of the underlying genetic mechanisms remain largely unknown. To investigate nondisjunction in mitotic and meiotic germ cells, we performed three-color FISH to detect numeric chromosome aberrations in testicular tissue samples from infertile men confirmed to have impaired spermatogenesis of unknown cause. FISH was employed to determine the rate of sex-chromosome aneuploidy in germ cells. Nuclei were distinguished as haploid or diploid, respectively. The overall incidence of sex-chromosome aneuploidy in germ cells was found to be significantly higher (P<.00001) in all three abnormal histopathologic patterns (range 39.0%-43.5%) as compared with normal controls (29.1%). The relative ratio of normal to aneuploid nuclei in the diploid cells of patients with impaired spermatogenesis was approximately 1.0, a >300% decrease when compared with the 4.42 ratio detected in patients with normal spermatogenesis. These results provide direct evidence of an increased incidence of sex-chromosome aneuploidy observed in germ cells of men with severely impaired spermatogenesis who might be candidates for ICSI with sperm obtained directly from the testis. The incidence of aneuploidy was significantly greater among the diploid nuclei, which suggests that chromosome instability is a result of altered genetic control during mitotic cell division and proliferation during spermatogenesis.

Prenatal diagnosis with use of fetal cells isolated from maternal blood: Five-color fluorescent in situ hybridization analysis on flow-sorted cells for chromosomes X, Y, 13, 18, and 21

OBJECTIVE: Currently, prenatal diagnosis of chromosome abnormalities requires invasive techniques such as amniocentesis and chorionic villus sampling that carry small but finite risks of fetal loss. A noninvasive approach is to isolate fetal cells from maternal blood by flow sorting followed by genetic interphase analysis with fluorescence in situ hybridization. Because the ratio of fetal to maternal cells is relatively low after flow sorting and to detect 90% to 95% of fetal aneuploidies associated with serious birth defects, a 5-color fluorescent in situ hybridization strategy is necessary for simultaneous detection of chromosomes X, Y, 13, 18, and 21 in all flow-sorted nuclei recovered from a specimen. STUDY DESIGN: Fetal nucleated red blood cells were isolated from maternal blood in 40 cases (10.4 to 27.0 weeks’ gestation) by flow cytometry on the basis of positive selection of CD71 + (transferrin receptor), CD45 –, and LDS751 staining. Each case was evaluated for 5-color fluorescent in situ hybridization efficiency by determining the percentage of flow-sorted nuclei containing 8 hybridization signals for chromosomes X, Y, 13, 18, and 21. RESULTS: A total of 42,312 flow-sorted nuclei from maternal blood samples were analyzed. In 5 of 16 (31%) cases with a male fetus, 0.16% of nuclei scored were identified as fetal by the presence of 1 signal each for chromosomes X and Y. Fetal trisomy 21 nuclei were accurately detected in 2 cases with a female fetus, each of which was subsequently confirmed. CONCLUSIONS: Five-color interphase fluorescent in situ hybridization analysis can be used to effectively analyze rare fetal aneuploid nuclei in enriched flow-sorted cells isolated from maternal blood. (Am J Obstet Gynecol 1998;179:203-9.)

Advantages and limitations of using fluorescence in situ hybridization for the detection of aneuploidy in interphase human cells

Fluorescence in situ hybridization with chromosome-specific DNA probes is being increasingly utilized for the detection of chromosome aberrations induced in vitro and in vivo by chemical and physical agents. Although potentially a powerful technique, FISH studies for aneupoloidy can be heavily influenced by cellular phenomena and hybridization artifacts which make the performance and interpretation of the results difficult. As a consequence, frequently hyperdiploid frequencies are reported in the literature which are substantially higher than one would expect based upon frequencies seen in conventional metaphase analyses. In this article, a number of the potential pitfalls that we have encountered while performing FISH analyses for aneuploidy are discussed and their potential impact on the observed hybridization frequencies is described. After considering these factors, the frequencies of lymphocyte nuclei containing 3 and 4 chromosome copies are compared between metaphase values obtained from published human population studies and interphase values obtained from similar studies using FISH. It is concluded that by using caution in the evaluation of slides, interphase studies using FISH to detect hyperdiploidy and polyploidy can provide estimates of numerical alterations which closely reflect those seen during metaphase analysis using either FISH or conventional approaches. However, due to the inability of interphase analysis to distinguish hyperdiploidy from polyploidy as well as other potential problems, frequencies of aneuploid nuclei obtained using single label FISH should only be considered approximations of absolute frequencies. For additional accuracy, multi-color FISH with two or more different probes shold be performed.

SepB: an Aspergillus nidulans gene involved in chromosome segregation and the initiation of cytokinesis.

In Aspergillus nidulans conidia, cytokinesis (septation) is delayed until three rounds of nuclear division have been completed. This has permitted the identification of essential genes that are involved in the coordination of cytokinesis with nuclear division. Conditional mutations in the sepB gene block septation but allow germinating spores to complete the first three rounds of nuclear division at restrictive temperature. sepB3 mutants demonstrate transient delays in M-phase, accumulate aneuploid nuclei and show defects in chromosome segregation. Molecular analysis of the sepB gene reveals that it is essential and possesses limited similarity to the CTF4 gene of Saccharomyces cerevisiae. Using temperature-shift analysis we show that sepB is required after the first nuclear division but before the onset of cytokinesis. A failure to execute the sepB function results in a block to nuclear division and leads to cell death at a time when wild-type cells would be undergoing cytokinesis. Finally, we demonstrate that sepB is also required for the uninucleate cell divisions of developing conidiophores. Our results suggest that sepB3 mutants accumulate specific nuclear defects that do not arrest mitosis, but block the initiation of septum formation. Thus, proper chromosome segregation and a functional sepB gene are required to initiate cytokinesis.

Impaired chromosome segregation in plant anaphase after moderate hypomethylation of DNA.

10(-6) M and 10(-5) M 5-azacytidine, demethylated around 9% and 17% of the 5-methylcytosine residues found in Allium cepa L. native DNA, respectively. Both treatments stimulated RNA synthesis in the cells of root meristems. On the other hand, the 10(-5) M treatment gave rise to multiple chromosomal anomalies in mitosis before any fall in the mitotic index was detectable, but no chromosomal breaks were ever seen. Serious lesions involved in chromatids and segregation in anaphase were preferentially found after hypomethylation of DNA sequences replicated in the second half of the previous S period: (i) sister telomeres remained unresolved at the cell equator while kinetochores had reached the poles, (ii) whole unsegregated chromosomes were pulled to one of the poles by obviously disfunctional kinetochores, resulting in an unbalanced distribution of chromatids, (iii) unsegregated chromosomes in other cells remained at the spindle equator as if kinetochores were nonfunctional, while cytoplasmic division took place before their migration to the poles. Frequently, a growing cytokinetic plate randomly cut the unsegregated chromosomes, giving rise to aneuploid nuclei. These anaphase failures are a firm basis to explain why the 10(-5) M treatment selectively depressed the rate of cell proliferation in these cells in the long run. On the other hand, if hypomethylation occurred at the first half of the previous S period, enlarged chromosomal segments were evident in most metaphases, while chromosome laggards and bridges were recorded in anaphase at rather similar frequencies after the different 5-azacytidine treatments. These data were consistently obtained both in the native mononucleate cells of meristems and in one subpopulation of synchronous cells labelled as binucleate by 5 mM caffeine.

Factors affecting formation of micronucleus-like structures after colchicine treatment of Trichoderma reesei

Micronucleus-like structures were produced in Trichoderma reesei only when 0.1% colchicine treatment was used to enhance nuclear division. The average DNA content of these 'small nuclei' was 30% that of the normal nuclei, indicating that they were aneuploid nuclei. Such small nuclei may be useful in transferring small amounts of DNA into protoplasts.

Chromosomal analysis in young vs. senescent human fibroblasts by fluorescence in situ hybridization: a selection hypothesis

Almost all previous studies on chromosomal analysis related to in vitro aging of human fibroblasts were done using only metaphase chromosomes. However, this procedure may provide only partial information since the aneuploidy presumably hidden in interphase cells would remain undetected. For this reason, we have analyzed aneuploidy both at interphase and at metaphase. Female (IMR-90) and male (IMR-91) cells were grown from the lowest to the highest population doubling levels and aneuploidy analysis was done by FISH with α-satellite DNA probes of 15 autosomes and two sex chromosomes. Our data on total aneuploidy in young cells indicate that significantly higher proportions of cells with aneuploidy can be detected at interphase than at metaphase. This presumably indicates that during active division of young cells, more aneuploid than diploid cells are selected against entry to mitosis. In contrast, interphase senescent cells from both strains show significantly fewer aneuploid nuclei than do young cells at interphase. This probably indicates that during senescence, there is greater selective pressure in the culture against long-term survival of aneuploid cells than against survival of diploid cells. Our study shows that cellular dynamics with respect to aneuploidy involving various chromosomes differs significantly at interphase and at mitosis during in vitro aging of human fibroblasts, and we propose a ‘Selection Hypothesis’ as an explanation to our findings.

Formation of a micronuclei‐like structure induced by colchicine treatment in Saccharomyces cerevisiae

AbstractMinute nuclei named “smaller nuclei” were generated when the cells of Saccharomyces cerevisiae were treated with colchicine. The formation of “smaller nuclei” seemed to be related to nuclear division because those nuclei were only produced under conditions suitable for nuclear division. The fact that the average DNA content of “smaller nuclei” was almost one tenth of that of the isolated normal diploid nuclei showed that the “smaller nuclei” are not condensed nuclei but aneuploid nuclei like micronuclei in animal cells. It appeared therefore likely that a micronuclei‐like structure could be produced by colchicine treatment in S. cerevisiae.

Flow-cytometric quantification in human gliomas of alpha satellite DNA sequences specific for chromosome 7 using fluorescence in situ hybridization.

Cell suspensions prepared from freshly frozen tissue specimens were used to examine aberrations in the number of chromosome 7 signals in 10 human gliomas. Nuclear DNA was hybridized in vitro with an alpha satellite DNA probe specific for the centromeric regions of chromosome 7, using fluorescence in situ hybridization (FISH). The intensity of the fluorescence signal from the hybridized probe was measured, together with the nuclear DNA content, by flow cytometry. The mean probe fluorescence of all nuclei was compared to the mean copy number per nucleus found with microscopic scoring. Moreover, the mean probe fluorescence ratio of DNA aneuploid nuclei relative to DNA diploid nuclei (FISHa/FISHd) was calculated to determine how the numerical aberration of chromosome 7 signals contributes to the DNA ploidy of the sample. The results from the flow-cytometric analysis and from microscopic evaluation were compatible. One of four tumors with DNA diploidy had a higher average intensity of FISH signal and a broader coefficient of variation in FISH signal than normal brain tissue; this was shown to be due to the gain of chromosome 7 signals. Although FISHa/FISHd correlated with DNA indices (P < 0.01), there were some disparities, probably due to other complex genotypic associations involving several gains or losses of chromosomes. Thus gain of chromosome 7 in gliomas is related to both DNA ploidy change and chromosome specific gain. It is concluded that flow-cytometric quantification of FISH is useful in investigating numerical aberrations of chromosomes and nuclear DNA content simultaneously.

Nuclear ploidy of normal and neoplastic hepatocytes from woodchuck hepatitis virus-infected and uninfected woodchucks

Flow cytometric analysis of the ploidy of normal and neoplastic hepatocyte nuclei obtained from adult woodchucks, a model of human hepadnavirus-induced hepatocellular carcinoma, was performed. All 36 samples of nuclei from non-neoplastic liver from woodchuck hepatitis virus-infected or uninfected liver were diploid, indicating that age-related nuclear polyploidization does not occur in this species, unlike other rodents. Individual or multiple hepatic neoplasms were obtained from each of 14 woodchuck hepatitis virus-infected woodchucks. Nineteen samples of hepatocellular carcinoma and eight adenomas were examined. Aneuploid nuclei were detected in 10 of the hepatocellular carcinomas and three of the adenoma samples. Similar DNA indexes, ranging from 1.11 to 1.22, were found in 7 of the 10 aneuploid HCCs and all 3 aneuploid adenomas. Nine of the 19 hepatocellular carcinoma samples and 5 of the 8 adenomas were diploid. Four of the diploid hepatocellular carcinomas had increased proportions of tetraploid nuclei. The presence of aneuploid nuclei was not related to histological appearance of the neoplasms or serum gamma-glutamyltranspeptidase levels. Because none of the hepatocellular carcinomas metastasized, the presence of aneuploidy could not be related to biological behavior. We determined the proportion of uninucleate and binucleate hepatocytes in hepatocellular carcinoma and nonneoplastic liver. Approximately 7% of hepatocytes were binucleate in nonneoplastic liver from woodchuck hepatitis virus-infected and uninfected liver. Only 2% of malignant hepatocytes were binucleate. The results of this study indicate that aneuploidy is a common change in hepatic neoplasms from woodchuck hepatitis virus-infected woodchucks.

DNA aneuploidy in Hodgkin's disease: a multiparameter flow cytometric analysis.

Paraffin-embedded lymph nodes from patients with Hodgkin's disease were examined for flow cytometric DNA content. In order to increase the sensitivity of the assay we tried to enrich for the neoplastic cells by bivariate analysis using a polyclonal anti-nucleolar antibody (AN-AB) and the forward scatter (FSC). DNA aneuploidy was found to be present in 67 of all 137 cases (49%), in 24 cases only demonstrable by dual-parameter analysis. The DNA index varied from 0.69 to 1.89 with a total of 22 hypo-diploid cases. The number of aneuploid nuclei exceeded the expected frequency of Reed-Sternberg (RS) and Hodgkin (H) cells in most of the analysed specimens. In conclusion, flow cytometry in Hodgkin's disease appears to give useful information regarding the ploidy status and evidence has been provided that the malignant cell population in Hodgkin's disease is not limited to the classical RS/H cells.

Tissue culture loss of aneuploid cells from carcinomas of the prostate.

The frequency of aneuploid cells in cultured prostate carcinoma specimens was investigated. Ploidy distribution of the original tissue was established by flow cytometry (FCM). Fluorescence in situ hybridization (FISH) of chromosome I was applied to directly isolated and cultured cells to investigate whether any modifications in the ploidy distribution of chromosome I took place during tissue culture. In six tumor specimens that were diploid by FCM and FISH, no differences were found in the ploidy distribution of chromosome I before and after tissue culture. In eight tumors that were aneuploid by FISH, the percentage of aneuploid nuclei was significantly reduced from 28.0 +/- 15.0 (range 13-59%) in uncultured cells to 9.1 +/- 4.4 (range 4-18%) after tissue culture. The reduction of aneuploid nuclei ranged from 44 to 85%, which means that the majority of the aneuploid cell populations that were observed in the original specimens were undetectable in cultured samples. This suggests a preferential growth of normal epithelial cells. The data presented can explain the high percentage of diploid karyotypes usually found in short-term cultured prostate carcinoma specimens.

High frequency of DNA ploidy abnormalities in preimplantation embryos of the rabbit.

The DNA ploidy of Feulgen-stained cell nuclei of in vivo preimplantation rabbit embryos was assayed by cytophotometry. DNA ploidy abnormalities were detected in single-cell nuclei readings by the criterion of > or = 5C DNA. These hypermodal DNA contents are referred as to DNA aneuploidy. Two, 4 and 6 days old rabbit embryos, all of normal gross morphology, were investigated. The incidence of embryos with DNA ploidy abnormalities increased from 17% in 2-day-old cleavage stages to 51% in 6-day-old expanded blastocysts. All these embryos were mosaics and the percentage of DNA aneuploid nuclei per embryo did not usually exceed 9%. Fifteen percent of the expanded blastocysts, however, contained up to 23% abnormal nuclei. Throughout the embryonic stages studied, the DNA content of abnormal nuclei was remarkably constant and averaged 5.8C. DNA aneuploid and euploid blastocysts did not differ in size. A maternal FSH treatment did not influence the DNA ploidy. This is the first report on the DNA ploidy pattern in preimplantation rabbit embryos. Our results indicate that DNA aneuploidy of single blastomeres is common in this species and occurs more often than generally assumed. The embryonic viability does not seem to be affected by the presence of DNA aneuploid blastomeres supporting earlier findings that a limited number of abnormal blastomeres is compatible with normal preimplantation development.

Behavior of Parental Genomes in the Hybrid Hordeum vulgare × H. bulbosum

In situ hybridization of labeled total genomic DNA with unlabeled blocking DNA enabled the parental origin of all chromosomes to be established in root tips of the mature sexual hybrid plant Hordeum vulgare x H. bulbosum. The parental genomes tended to remain spatially separated throughout the cell cycle, with the chromosomes of H. vulgare origin lying in a more central domain than those of H. bulbosum origin. During anaphase and telophase, chromosomes of H. bulbosum origin tended to lag. Although the chromatids usually separated, they did not have the V shape characteristic of anaphase chromatids. Aneuploid nuclei, missing H. bulbosum origin chromosomes, arose when the lagging chromatids were not incorporated into the daughter nuclei, although most cells remained diploid. Some interphase cells contained micronuclei, all of which were of H. bulbosum origin. Information about chromosome disposition and movement is important to enable the understanding of chromosome stability.

Multiparameter Flow Cytometric Analysis of Neoplasms of the Central Nervous System: Correlation of Nuclear Antigen p105 and DNA Content with Clinical Behavior

Analysis of the DNA content of various solid tumors and hematological malignancies may provide useful prognostic information. To date, however, there has been a striking lack of correlation between DNA content in neoplasms of the central nervous system and clinical behavior. Simultaneous quantitation of DNA content and proliferation-associated nuclear antigen (p105) by flow cytometry was performed on paraffin-embedded tissues representing three major groups of central nervous system neoplasms--1) 21 astrocytic tumors, 2) 13 pituitary tumors, and 3) 19 meningiomas--and the results were correlated with clinical behavior. All 4 well-differentiated gliomas were diploid, while 3 of 9 anaplastic astrocytomas and 1 of 8 glioblastomas had a demonstrable aneuploid peak. Three of 13 pituitary tumors had an identifiable aneuploid peak, while only 2 of 19 meningiomas had an aneuploid DNA content. Cell-cycle analysis of the malignant gliomas revealed a significantly higher proliferative index (PI, %S + G2M) compared with the well-differentiated astrocytomas (P less than 0.05). Within the subgroup of diploid anaplastic astrocytomas, however, extended patient survival appeared to be associated with a higher PI. For diploid pituitary adenomas, the PI was consistently lower in the 3 tumors that recurred than it was in the remaining 8 adenomas. Nuclear antigen quantitation of diploid tumors showed a wide range of p105 expression in G0G1 cells, suggesting that, within each tumor, the cells are heterogeneous with respect to proliferative activity. Aneuploid nuclei of glial tumors showed enhanced expression of p105 relative to diploid cells of the same specimen. In pituitary tumors, the median G2M/G0G1 fluorescence ratio for p105 was significantly higher (P less than 0.05) for the 3 diploid recurrent tumors than for those that did not recur. These data support the assumption that the aggressive clinical course of malignant glial neoplasms may be related to an abnormal DNA stemline and/or an alteration in cell proliferative activity. Cell cycle analysis and measurement of p105 by this technique may provide information useful from both a prognostic standpoint and in directing adjuvant therapy.