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γ-secretase inhibitors have been proposed as drugs able to kill cancer cells by targeting the NOTCH pathway. Here, we investigated two of such inhibitors, the Benzyloxicarbonyl-Leu-Leu-Nle-CHO (LLNle) and the N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), to assess whether they were effective in killing human glioblastoma tumor–initiating cells (GBM TIC) in vitro. We found that only LLNle was able at the micromolar range to induce the death of GBM TICs by apoptosis. To determine the cellular processes that were activated in GBM TICs by treatment with LLNle, we analyzed the amount of the NOTCH intracellular domain and the gene expression profiles following treatment with LLNle, DAPT, and DMSO (vehicle). We found that LLNIe, beside inhibiting the generation of the NOTCH intracellular domain, also induces proteasome inhibition, proteolytic stress, and mitotic arrest in these cells by repressing genes required for DNA synthesis and mitotic progression and by activating genes acting as mitotic inhibitors. DNA content flow cytometry clearly showed that cells treated with LLNle undergo arrest in the G2-M phases of the cell cycle. We also found that DAPT and L-685,458, another selective Notch inhibitor, were unable to kill GBM TICs, whereas lactacystin, a pure proteasome inhibitor, was effective although at a much less extent than LLNle. These data show that LLNle kills GBM TIC cells by inhibiting the proteasome activity. We suggest that LLNle, being able to target two relevant pathways for GBM TIC survival, may have a potential therapeutic value that deserves further investigation in animal models. (Mol Cancer Res 2009;7(11):1822–34)

In order to better understand the relationship of DNA ploidy, dysplasia, early cancer, and colorectal tumor progression, 11 colorectal adenomas containing carcinoma invading the submucosa were investigated using DNA flow cytometry. Multiple frozen samples were taken from the selected sectors corresponding to adenoma tissue with low-grade dysplasia, high grade dysplasia and early cancer. Sampling accuracy was performed under histologic examination by multiple cryostatic sections. Data were compared with previously reported results in non-cancerous adenomas and advanced carcinomas. Incidence of DNA aneuploidy among the dysplastic regions of the adenomas containing carcinomas resulted higher than that observed in non-cancerous adenomas (p=0.02). Furthermore, among the DNA aneuploid populations, the frequency of clones with high DNA Index (DI>1.3) was slightly higher in adenomas with cancer than in adenomas without cancer (p=0.07). We suggest that differences may exist in DNA aneuploidy evolution between these two types of lesions. In early cancer, the near-diploid clones were 57% with respect to 18% (p=0.01) in advanced cancer since in this latter case the majority of the DNA abnormal clones were in the near-hypertriploid region (82%). Thus, the acquisition of the invasive phenotype appears to be linked with the expansion and stabilization of high DNA aneuploid clones. Further analysis on a larger number of cases of adenomas containing carcinoma are necessary to validate these interpretations.

We have recently described a novel nuclear antigen, AF-2, which is related to cell cycle dependent alterations of chromatin structure. We show by two parameter flow cytometry on a cell by cell basis that the antigen is accessible to specific monoclonal antibodies only in mitotic and postmitotic early G1-phase cells. The evaluation of nuclease susceptibility and AF-2 antigen accessibility reveals different subcompartments of the G1 -phase of the cell cycle with distinct chromatin conformations. Digestion with DNase I seems to alter the chromatin structure according to concentration and this is reflected by an increase of the antigen accessibility. Chromatin in the more condensed early G1-phase is specifically digested by lower concentrations of the enzyme than chromatin in later stages of interphase. Chromatin from cells in the late-G1, S-, and G2-phases shows a higher relative resistance to DNase I and a reduced accessibility of the AF-2 antigen to monoclonal antibodies. Nuclease S1 has a similar effect on chromatin topology, as revealed by the reaction with anti-AF-2 antibodies, without digestion of detectable amounts of DNA. The antigen becomes available to the antibodies in almost all cells by digestion with high concentrations of DNase I or Nuclease S1.

The relationship between survival and flow cytometric DNA-ploidy and other prognostic factors such as histological subtype, anatomical tumor site, patient sex and age was investigated in 153 patients with intracranial neuroepithelial tumors who underwent surgical treatment. We found a trend toward poorer survival from anaplastic astrocytomas and glioblastomas with respect to low-grade (I and II) astrocytomas (which did not differ significantly); accordingly, patients were grouped into these 3 histologic subgroups. Thirty-seven of the 153 tumors (24.2%) were aneuploid with a median DNA-index (DI) of 1.3 (range: 1.2-2.0). DNA-ploidy correlated with histology, since anaplastic astrocytomas and glioblastomas were significantly (p = 0.041) more frequently aneuploid (around 30%) than low-grade astrocytomas (around 10%). Patients with DNA-aneuploid tumors (i.e., with DI not equal to 1.00) survived for a shorter time (31.4 weeks) than patients with DNA diploid tumors (75.1 weeks) (p less than 0.001). This difference was confirmed by Cox's multivariate analysis. Aneuploid tumors were associated with a poorer survival (p = .0002) when compared with diploid tumors, resulting in a relative risk point estimate (RR) of 2.41, 95% confidence interval (Cl) = 1.55-3.74. Histological subtype was also significantly associated with survival (p less than 0.0001), with RRs of 2.09, 95% Cl = 1.13-3.86 and 3.59, 95% Cl = 1.96-6.59 for anaplastic astrocytomas and glioblastomas, respectively, compared to low-grade astrocytomas. We therefore suggest that the flow cytometric measurement of DNA-ploidy has relevant significance in predicting survival in patients treated for intracranial neuroepithelial tumors.

The aim of this study was to analyze by flow cytometry the effect of cis-diamminedichloroplatinum II (CDDP) and retinoic acid (RA) on the cell cycle of a neuroblastoma cell line (SK-N-BE (2)C NB) and to correlate the kinetic data with cell morphology. CDDP at 1 microgram/ml induced a dramatic G2 + M cell cycle phases block (nearly 200% increase with respect to control) 2 days after treatment. The G2 + M block was spontaneously reversed starting from the 4th day. The cells treated with 10 microM RA were, instead, induced to irreversibly enter the G0 + G1 phase of the cell cycle (nearly 20% increase with respect to control) 48 h after treatment. Neurite-like structures were observed for both CDDP and RA treated cells. These data suggest different cell cycle dependent molecular mechanisms and different degrees of differentiation during CDDP or RA treatment of NB cells.

The molecular pathways and the timing of genetic events during human colorectal carcinogenesis are still not fully understood. We have addressed the intratumor heterogeneity of the mutational status of the k‐ras oncogene and of the p53 oncosuppressor gene during the adenoma–carcinoma sequence by investigating 26 human colorectal adenomas containing early cancer. An intratumor comparative analysis was obtained among the adenomatous and carcinomatous component pairs. Additionally, we have analyzed 17 adenomas having cancer in the near vicinity. The adenomatous components of the adenomas containing early cancer and the adenomas having cancer in the near vicinity had comparable frequencies for k‐ras mutations (28 and 47%) but different for p53 mutations (52 and 7%,p‐value = 0.01). Interestingly, the adenomatous and carcinomatous components of the adenomas containing early cancer were rarely heterogeneous for the k‐ras mutational status (only in 13% of the cases) but were characterized by heterogeneity of the p53 status in 59% of the cases (p‐value < 0.01). In addition, the mutations of p53 for the adenomatous components of the adenomas containing early cancer were statistically significantly associated with severe dysplasia (p-value = 0.01). Intratumor homogeneity of k‐ras status during the human colorectal adenoma–carcinoma sequence suggests that the role of k‐ras is more related to tumor initiation than to tumor progression. On the contrary, intratumor heterogeneity of p53 mutations indicates that the type of the p53 mutations may also be relevant for selection and expansion of new subclones leading to tumor progression.

BACKGROUND & AIMS: Deletions at chromosome 1p have been observed frequently in human colorectal adenocarcinomas, suggesting that loss of genes in this chromosome arm is relevant for tumorigenesis. The aim of this study was to investigate whether 1p deletions are already present in adenomas within selected foci of dysplasia and early cancer using two-color fluorescence in situ hybridization. METHODS: Fifty-one sectors characterized by low- and high-grade dysplasia and early cancer were microdissected from 34 adenomas, and isolated epithelial nuclei were subjected to hybridization with probes to the telomeric and centromeric regions of chromosome 1. RESULTS: Deletions of 1p were detected in 13 of 34 adenomas (38%). In particular, low/moderate and high dysplasia and foci of early cancer had 1p deletion frequencies of 31%, 44%, and 50%, respectively. CONCLUSIONS: Compared with classic cytogenetics, fluorescence in situ hybridization seems to be a particularly useful methodology to detect 1p deletions in human colorectal adenomas. The present findings indicate that loss of genes from the 1p chromosome arm may play an important role during the early steps of the colorectal carcinogenesis. (Gastroenterology 1996 Jul;111(1):102-7)

Background Most patients affected by Glioblastoma multiforme (GBM, grade IV glioma) experience a recurrence of the disease because of the spreading of tumor cells beyond surgical boundaries. Unveiling mechanisms causing this process is a logic goal to impair the killing capacity of GBM cells by molecular targeting. We noticed that our long-term GBM cultures, established from different patients, may display two categories/types of growth behavior in an orthotopic xenograft model: expansion of the tumor mass and formation of tumor branches/nodules (nodular like, NL-type) or highly diffuse single tumor cell infiltration (HD-type). Methods We determined by DNA microarrays the gene expression profiles of three NL-type and three HD-type long-term GBM cultures. Subsequently, individual genes with different expression levels between the two groups were identified using Significance Analysis of Microarrays (SAM). Real time RT-PCR, immunofluorescence and immunoblot analyses, were performed for a selected subgroup of regulated gene products to confirm the results obtained by the expression analysis. Results Here, we report the identification of a set of 34 differentially expressed genes in the two types of GBM cultures. Twenty-three of these genes encode for proteins localized to the plasma membrane and 9 of these for proteins are involved in the process of cell adhesion. Conclusions This study suggests the participation in the diffuse infiltrative/invasive process of GBM cells within the CNS of a novel set of genes coding for membrane-associated proteins, which should be thus susceptible to an inhibition strategy by specific targeting. Massimiliano Monticone and Antonio Daga contributed equally to this work

A model on the origin and evolution of DNA aneuploidy based on a postulated mechanism of DNA asymmetrical cell division is presented. Asymmetry in cell division would be at the origin of hypodiploid cells in the near-diploid region. Tetraploidization of a hypodiploid cell would be one of the main routes by which advanced tumors may evolve to aneuploidy in the near-triploid region. The model is supported by nuclear DNA content data obtained by high resolution flow cytometry from fresh/frozen material during the colorectal tumor progression.

Aim of the study was to evaluate the relationship between the mitogenic stimulus interleukin-3 to normal murine mast cells and the cell cycle dependent expression of the nuclear c-myc protein. In order to do that on a cell by cell basis, we measured the nuclear c-myc protein simultaneously by flow cytometry, via specific monoclonal antibodies, and the DNA content via the intercalating dye propidium iodide. When cells were deprived from interleukin-3 (IL-3), proliferation was inhibited and the majority of cells arrested in early G1 (G1A, characterized by low c-myc content). Readdition of IL-3 resulted in a slow transition of cells from G1A to late G1 (G1B, at higher c-myc content) before DNA synthesis started. G1A cells with low c-myc content do not undertake DNA synthesis. Using a stathmokinetic methodology we confirmed that the G1A cells are early postmitotic G1 phase cells. The low content of c-myc within these cells appears a direct consequence of reduced c-myc levels during mitosis. Cumulatively, the data suggest that c-myc protein levels of murine mast cells fall at mitosis and that these levels must rise before cells can traverse the G1 phase. Our data are compatible with a model in which c-myc protein content of G1 phase cells has to reach a critical threshold before the cells can move further into the cell cycle.

Progressive in vitro culturing of interleukin-3 (IL-3) dependent normal murine mastocytes (PB-3) resulted in a variant cell line (PB-1) able to grow without exogenous IL-3 and which was tumorogenic in syngenic mice. Bivariate flow cytometry was used to evaluate the c-myc protein and DNA content of PB-3 and PB-1 cells. The c-myc protein was detected by specific monoclonal antibodies. Kinetic characteristics of PB-3 and PB-1 cell lines, namely, the duration of the G1, S and G2 + M cell cycle phases were also evaluated using the bromodeoxyuridine (BrdU) pulse-chase method and BrdU/DNA flow cytometry. Levels of c-myc protein in PB-1 cells were about two-fold higher than those of PB-3 cells in all cell cycle phases. Mean duration of the cell cycle (Tc) was 15.3 h for PB-3 cells and 12.4 h for PB-1 cells. Shortening in Tc for the transformed cells was due to a decrease of nearly 30% in mean duration of the G1 phase (from 8 h to 5.7 h). No significant differences were found in the duration of the S and G2 + M phases. These results indicate that acquired IL-3 independency in vitro and tumorogenicity of PB-1 cells were accompanied by a doubling of c-myc protein level and by a parallel shortening, or bypass, of the regulatory events within the G1 phase of the cell cycle.

The cell cycle kinetic characteristics of chick endochondral chondrocytes differentiating in vitro were studied by flow cytometry. In addition, the synthesis of type I and type X collagens of the same cells was evaluated by immunoprecipitation. Dedifferentiated cells, derived from chick embryo tibiae and grown attached to a substratum, were characterized by type I collagen synthesis, a high growth fraction (GF = 0.94), minimal cell loss factor (phi = 0.02), and a total cell cycle time of the proliferating cells of about 17 h (tG1 = 8 h, tS = 5 h, and tG2 + M = 4 h). Transfer of dedifferentiated cells to suspension culture on agarose-coated dishes induced differentiation to hypertrophic chondrocytes. These were characterized by type X collagen synthesis, a low growth fraction (GF = 0.52), maximal cell loss factor (phi = 1.0), and a total cell cycle time of the proliferating cells of about 73 h (tG1 = 53 h, tS = 12 h, and tG2 + M = 8 h). The transition from dedifferentiated chondrocytes to hypertrophic chondrocytes was accompanied by large increases of the duration of all the cell cycle phases and of the number of quiescent and degenerating cells. Associated with these alterations in cell cycle kinetics was a switch from type I to type X collagen synthesis. Further preliminary data suggest that the population of differentiating chondrocytes (a state between dedifferentiated and hypertrophic chondrocytes) comprises a heterogeneous population of fast and slow growing cells.

A new flow cytometric method combining light scattering measurements, detection of bromodeoxyuridine (BrdU) incorporation via fluorescent antibody, and quantitation of cellular DNA content by propidium iodide (PI) allows identification of additional compartments in the cell cycle. Thus, while cell staining with BrdU-antibodies and PI reveals the G1, S, and G2 + M phases of the cell cycle, differences in light scattering allow separation of G2 phase cells from M phase cells and subdivision of G1 phase into two compartments, i.e., G1A representing postmitotic cells which mature to G1B cells ready to initiate DNA synthesis. The method involves fixation of cells in 70% ethanol, extraction of histones with HC1, and thermal denaturation of DNA. This treatment appears to enhance the differences in chromatin structure of cells in the various phases of the cell cycle to the extent that cells could be separated on the basis of the 90 degrees scatter. Mitotic cells show much lower scatter than G2 phase cells, and G1 postmitotic cells (G1A) show lower scatter than G1 cells about to enter the S phase (G1B). Light scattering is correlated with chromatin condensation, as judged by microscopic evaluation of cells sorted on the basis of light scatter. The method has the advantage over the parental BrdU/DNA bivariate analysis in allowing the G2 and M phases of the cell cycle to be separated and the G1 phase to be analyzed in more detail. The method may also allow separation of unlabeled S phase cells from mitotic cells and distinguish between labeled and unlabeled mitotic cells.

Cell cycle synchronization of MCF-7 hormone-sensitive human breast cancer cells has been evaluated after sequential treatment with tamoxifen and 17 beta-estradiol. The analysis was performed by flow cytometry. Two methods were used, one for single-parameter DNA content analysis, and one for bivariate analysis of DNA content and amount of incorporated bromodeoxyuridine (BrdUrd) into DNA using a specific monoclonal antibody. According to the BrdUrd method, tamoxifen was found (over a 30h period) to decrease (with respect to cells grown in control medium) the fraction of cells in S phase from 45% to 20%, to increase cells in G0 + G1 from 47% to 68%, and to induce a slight build-up of cells in G2 + M. Subsequent addition of estradiol resulted in partial synchronous recruitment of the cells from G0 + G1 to progress through the S phase; after 6-8 h delay time, the percentage of cells in G0 + G1 decreased by 50% and cells in S increased by 175%. The bivariate BrdUrd technique offered more reliable and detailed information than the single-parameter DNA analysis for differentiating and measuring the time course of estrogen-recruited cells as they progressed through early and late S phase, and has the potential for a very detailed cell kinetic analysis of both in vitro and in vivo hormone-sensitive cells.

A human cell system suitable for serving as a biological dosimeter should show high specificity that is ideally only responding in a quantitative and reproducible manner to the dose received. If high resolution image analysis is chosen as the method forr detecting such changes, functional or preparatory influences on the image of the target cells should be well known to control them suitably. In the present study the Feulgen staining procedure was chosen to study such possible influences on the nuclear chromatin structure using mouse L fibroblasts as a model cell population. In order to investigate the variability primarily due to functional heterogeneity, the Feulgen hydrolysis behaviour of GO, Gl, S and G2 cells was studied. It could be shown that depoly-merization and depurination slightly depend on the cellular phase in the cycle, indicating a different acid stability of the chromatin matrix. It could further be shown that other preparatory conditions as, for example, the way of spreading the cells or the conventional trypsinization are critically influencing the features extracted from the nuclear images. The results obtained so far will be used to develop a strategy for an optimum selection and preparation of human cells for dosimetric purposes using high resolution image analysis.

Cell cycle variations and DNA aneuploidy, were investigated in different phases of azoxymethane (AOM)-induced colon carcinogenesis in rats by flow cytometry. K-ras gene mutations (transitions Gright curved arrow A) were frequently detected in aberrant crypt foci (ACF) initial pre-neoplastic lesions. The fraction of cells in the G2M-phase of the cell cycle was higher in ACF compared to the normal mucosa of control rats. A similar modification of the cell cycle was found in adenomas and adenocarcinomas but, unexpectedly, also in morphologically normal mucosa from AOM-treated animals indicating that AOM treatment permanently modifies cell cycle control in rat colon mucosa. These alterations, however, were not associated with DNA aneuploidy as reported in human sporadic colorectal cancer, suggesting that tumour development in AOM-treated rats is less dependent on aneuploidy.

We have recently described a novel protein (AF-2), conserved between fission yeast and man, and we have shown by flow cytometry (FCM) that AF-2 is highly accessible to specific monoclonal antibodies (MoAbs) in mitotic and postmitotic early-G1 phase cells. The aim of the present study was to optimize the FCM methodology using MoAbs against AF-2 and to show that the evaluation of the mitotic cells, using different cell lines, was quantitative and reproducible. We found that a method based on fixation with ethanol, instead of formalin, resulted in improved DNA histogram coefficients of variation and implemented separation of early-G1 cells from late-G1 cells. In addition, by eliminating several cell permeabilization and protein salt extraction steps, the method became straightforward, conserved a clear-cut separation of the green fluorescence of M- with respect to G2-phase cells, and did not significantly affect cellular integrity. The coefficient of correlation among the mitotic index values evaluated by this FCM method using MoAbs against AF-2 and by microscopic visual counting was R = 0.94. When the FCM/AF-2 method was tested against an independent FCM method, which allows clear separation of M- and G2-phase cells according to 90 degrees scattering, we found R = 0.93. We conclude that MoAbs against the AF-2 protein may be used in FCM for quantitative analysis and for isolation of M-phase cells, providing as well, the identification of the early-G1 cell subcompartment. The method may, in addition, be useful for the simultaneous detection of cytoplasmic cytokeratin and nuclear AF-2 antigen.