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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.

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.

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.

An International “DNA Cytometry Consensus Conference” was held in October 1992, in Maine (USA): The Author reports the opinions concerning bladder tumours, expressed by the experts at the Conference. Models for future studies with Flow Cytometry are also reported.

Mutant ras genes occur frequently in human neoplasia and, in particular, in pancreatic, colorectal, and lung adenocarcinomas. Recent evidence suggests that G--T and G--C transversions of the Ki-ras gene in codon 12 may lead to biological effects in vitro and in vivo that may be associated with an abnormal cell cycle and increased tumour aggressiveness. The role of Ki-ras activation (a G--C transversion in codon 12, arginine for glycine) in the cell cycle and apoptosis was investigated using control and permanently transfected NIH3T3 mouse fibroblasts. Flow cytometry was used to evaluate the G1-, S- and G2M-phase transit times, the potential doubling time, the growth fraction, and the cell loss factor during asynchronous exponential growth. Apoptosis was induced in both cell lines by absence of growth factors for an extended period of time (72 h) and quantitatively evaluated using the TUNEL method coupled with flow cytometry. It was found that codon 12 G--C Ki-ras transfected cells compared with controls, had a significant prolongation of G1 by about 50%, a reduction of the G2M transit time by 30%, and a decrease of the cell loss factor by about 90%. Apoptotic cells were about 10% in control and less than 0.5% in Ki-ras transfected cells after 72 h starvation-confluency. These data suggest that codon 12 G--C Ki-ras activation in mouse NIH3T3 fibroblasts is associated with deregulation of checkpoint controls in the G1 and G2M phases of the cell cycle and inhibition of apoptosis. It appears plausible that these cell mechanisms are related to a proliferative advantage and that they may also be important in the progression of human tumours characterized by specific Ki-ras mutations.

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.

Guidelines are given to assist the standardisation of DNA flow cytometry in clinical pathology. They have been agreed by a group of twelve scientists from nine European countries.

The aim of this pilot study was to explore the prognostic relevance of cell kinetics parameters on the local control of patients affected by head and neck squamous cell carcinoma (HN-SCC), randomly assigned to receive either alternating chemoradiotherapy or partly accelerated radiotherapy. Between 1992 and 1995, 40 patients with HN-SCC at stages III and IV entered the study. Multiple primary tumor biopsies were obtained 6 h after in vivo infusion of bromodeoxyuridine, an analogue of thymidine that is incorporated in DNA-synthesizing cells. In vivo S-phase fraction labeling index (LI), duration of S-phase (TS), and potential doubling time (Tpot) were obtained by analysis of the flow cytometric content of bromodeoxyuridine and DNA. Twenty patients were treated by alternating chemotherapy and conventional radiotherapy (arm A), whereas 20 other matching patients received partly accelerated radiotherapy alone (arm B). Univariate local control analysis showed that LI, TS, and Tpot were not prognostically significant in either arm. However, local control probability at 2 years for fast growing tumors, characterized by a LI of 9%, was higher for patients treated with alternating chemoradiotherapy than it was for those treated with partly accelerated radiotherapy alone (68 versus 39%). Conversely, local control probabilities for slow proliferating tumors (LI,9%) treated in the two arms were similar. These results suggest a potential role for alternating chemotherapy and radiotherapy in HN-SCC patients with fast growing tumors.

The purpose of the study was to determine whether pretreatment tumor cell kinetics can predict local control in elderly patients affected by squamous cell carcinoma of the head and neck (SCC-HN) and help guide different therapeutic modalities. Over a 6-year period, 52 patients with stage II to IV SCC-HN and aged more than 70 years were given an infusion of bromodeoxyuridine (BrdUrd) 6 hours prior to tumor biopsy sampling. The simultaneous labeling S phase fraction (LI) and duration (Ts) as well as potential doubling time (Tpot) were measured with flow cytometric analysis of BrdUrd and DNA content. Patients were then treated as follows: 14 with conventional radiotherapy; 13 with partly accelerated radiotherapy; 11 with chemoradiotherapy; 14 with surgery plus adjuvant radiotherapy. Univariate analysis showed that, independently of treatment type, patients with fast growing SCCs-HN characterized by Tpot valueor = 5 days had a lower three-year local control than patients with slow growing tumors with Tpot value5 days. Our results also demonstrated that surgery or chemoradiotherapy were effective treatments for fast growing tumors. Radiotherapy alone, instead, was more effective for slow growing tumors. Our data suggest that in vivo cell kinetics may play a role as additional prognostic factor for elderly patients with SCC-HN and predict the outcome of different treatments.

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)

Detailed information about intratumor K-ras2 mutations in colorectal adenocarcinomas and a possible association with DNA content heterogeneity is still lacking. DNA diploid and aneuploid subclones, detected among multiple histologically selected primary sectors (57 superficial and 40 deep) and 9 lymph node metastases, were flow cytometrically sorted and separately submitted to codons 12-13 K-ras2 mutation spectrum analysis. DNA aneuploidy was absent among 20 near and 20 distant mucosa sites and present in 7/9 lymph node metastases and in 17/19 primary tumors (90%). Primary intratumor DNA multiclonality was approximately 50%. Degree of DNA aneuploidy (DNA Index) distribution was nonrandom and showed peaks at approximate mean DNA Index values 1.2, 1.5, and 1.8. K-ras2 mutations were detected in 0/20 mucosa cases, in 2/9 lymph node metastases, and in 9/19 adenocarcinomas (47%). No more than one mutation type per tumor was detected. Intratumor distribution of K-ras2 mutations was homogeneous in 6 and heterogeneous in 3 cases. Homogeneous distribution was associated with DNA near-diploid aneuploidy. K-ras2 mutations were strongly associated with DNA Index in the near-diploid region (83%) and almost absent (5%) among DNA near-triploid subclones (P = 0.0001). K-ras2 mutation intratumor heterogeneity indicates that sampling of the tumor may be a critical step and suggests that K-ras2 activation may be a late event in a subgroup of tumors. Our data also suggest the existence of an early process of the colorectal carcinogenesis that favors both K-ras2 mutations and DNA near-diploid aneuploidy. Onset of DNA near-triploid subclones appears, instead, to be independent from K-ras2 activation.

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

Extensive chromosome and DNA content heterogeneity within and between human solid tumors has been observed using both classical karyotype and DNA cytometry. Experimental evidence suggests, at least in some tumor types, that DNA stemline heterogeneity in tumor progression is according to a three-compartment model with diploidy shifting to tetraploidy and then to hypotetraploidy.The human colorectal adenoma-carcinoma sequence appears as one of the most potentially informative systems for the study of DNA stemline heterogeneity in human tumors since adenomas, adenomas with early cancer, and adenocarcinomas in nontreated patients represent clear morphologically distinct stages of tumor progression. The quantitative measurement of DNA content in the G0.1 phase of the cell cycle was performed by high resolution flow cytometry in a large number of cases using multiple fresh or frozen samples.The distribution of the degree of DNA aneuploidy values, also known as DNA index, (DI not equal to 1) among 467 human precancer and cancer colorectal lesions was clearly nonrandom and showed modes at DI = 0.9, 1.2, 1.5, 1.8, and 2.2 with a clear valley at DI = 1.3. Whereas DNA aneuploid subclones within early lesions were up to about 80% near-diploid (DIor = 1.3), DNA subclones within advanced cancer were in the vast majority with DI = 1.5-1.8 and, in a small fraction, with DI2. In addition, in adenomas with early cancer, which represent a link in colorectal tumor progression, early and late DNA stemlines often coexisted.The natural history of the colorectal adenoma-carcinoma sequence appears to be characterized by near-diploid subclones as early events and by late-stage hypotetraploidy. A new model is proposed that predicts the origin of the near-diploid subclones by "loss of symmetry" in cell division and their evolution (in particular hypodiploid) to the late-stage hypotetraploidy by tetraploidization. This model agrees with recent data associating molecular biology events, cytogenetic data, and DNA stemline heterogeneity in colorectal and other tumor systems.

Cell cycle and cell population characteristics, as obtained for head and neck cancers by flow-cytometry analysis of a single tumor sample, after in vivo bromodeoxyuridine (Burd) infusion, were proposed for their prognostic value and for their potentials for radiotherapy planning (conventional vs accelerated) and monitoring. DNA ploidy, the S phase fraction labeling index (LI), and duration (Ts) as well as cell population potential doubling time (Tpot) were measured in 42 head and neck squamous cell carcinoma patients and analyzed along with histopathological and clinical data. Twenty-seven patients received standard radiotherapy (70 Gy/35 fractions/7 weeks) whereas 15 patients were treated with the concomitant boost technique (75 Gy/40 fr/6 weeks). The univariate analysis of 31 patients, for whom all the cell kinetic parameters were available, indicated that local control probability was strongly affected by lymph node status (p = 0.05) and by potential doubling time (p = 0.04). Patients having tumor Tpot5 days had markedly lower two-year local control rate (13%) than patients with Tpot5 days (68%). Furthermore, tumors with Tpotor = 5 days exhibited a trend toward better local control after concomitant boost regimen compared with the patients treated with standard regimen (p = 0.06). These preliminary results point out that Tpot could play a role as additional prognostic factor influencing disease outcome in head and neck carcinomas treated by radiotherapy. In patients with fast growth-rate tumors (Tpotor = 5 days) a more aggressive radiotherapy combination or chemo-radiotherapy should be suggested.

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.

Exposure of human promyelocytic leukemic HL-60 cells to the topoisomerase I inhibitor camptothecin (CAM) triggers endonucleolytic activity and apoptotic death of these cells. The nucleolytic effect is seen 2-4 h after drug addition and is highly selective to cells progressing through S phase. Concomitant with degradation of DNA, which is preferential to the nucleosomal DNA linker sections, extensive proteolysis takes place in these cells. Cellular RNA, however, is initially degraded to a much lesser degree than DNA or protein. Both endonucleolysis and proteolysis triggered by CAM in S-phase HL-60 cells can be prevented by the protease inhibitors N-tosyl-L-phenylalanylchloromethyl ketone (TPCK), N-tosyl-L-lysylchloromethyl ketone (TLCK) or partly by N-tosyl-L-arginine methyl ester (TAME), added simultaneously with CAM, or up to 30 min after exposure to CAM, at their respective concentrations known to inhibit proteases. The protective effect of these protease inhibitors on DNA degradation cannot be due to the suppression of cell progression through S phase because cells still replicate DNA in their presence, albeit at a reduced rate. Furthermore, TPCK and TLCK protect rat thymocytes against endonucleolysis induced by prednisolone. In the latter cell system, (considered a classic model of apoptosis), endonucleolysis, which primarily affects G0/G1 cells, is unrelated to cell progression through S phase. The present data suggest that the endonucleolysis and proteolysis which accompany apoptotic cell death are coupled, and the proteolytic step is needed for DNA degradation to occur.

Cell kinetic parameters were evaluated using the method based on in vivo incorporation of Bromodeoxyuridine (BrdU) and flow cytometric (FCM) analysis in 30 human epidermoid head and neck tumors from oropharynx, oral cavity, rhinopharynx, larynx and lips. BrdU was injected four/six hours before the obtainment of multiple bioptic samples from the tumor tissues. The flow cytometric method was carried out on 70% ethanol fixed cell suspensions based on established protocol for the simultaneous evaluation of DNA content and BrdU uptake using anti-BrdU monoclonal antibodies. We have evaluated the following FCM parameters: DNA ploidy, the degree of DNA aneuploidy (DNA index), Labelling Index (LI), duration of s-phase (Ts) and tumor potential doubling time (Tpot). LI values ranged from 1.5 to 20% with a median value of 10%. The median LI of DNA diploid tumors was 5.4% compared to 14% in DNA aneuploid tumors. Ts values ranged from 8 to 11, the median value being 10 hours. Tpot values ranged from 2 days to 16 days, the median Tpot being 5 days. The large heterogeneity of all these parameters indicates that these tumors may have a different degree of biologic aggressiveness (9). Tpot values did not correlate with DNA ploidy nor with lymph node metastasis status. Tpot values did not correlate in a statistically significant manner with degree of differentiation although shorter Tpot were more frequently observed in moderate or poorly differentiated tumors. Our study shows that the FCM-BrdU technique in vivo is feasible in a clinical setting to evaluate the proliferative behaviour of head and neck tumors, before any specific therapeutic decision is taken after surgery is performed. It is likely that tumors with more aggressive biological behavior, as indicated by LI15%, DNA aneuploidy and Tpot5 days, may benefit from more aggressive therapies such as accelerated regimeus of radiotherapy and/or other multimodal therapies in respect to tumors with slow growth rate (LI15%), DNA diploidy and Tpot5 days. So far, however, it still remains to be demonstrated from randomized clinical trials if the knowledge of such individualized cell Kinetic parameters really can help to choose the most effective therapy for every individual patient.

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.

Rat thymocytes were treated in culture with prednisolone or the DNA topoisomerase I or II inhibitors, camptothecin (CAM) or teniposide (TN), and proportions of cells in different phases of the cell cycle were estimated by flow cytometry using a staining methodology which makes it possible to discriminate between G0 and G1 cells, as well as to recognize the cells which undergo apoptosis. The appearance of apoptotic cells in cultures treated with pharmacological concentrations of these drugs, observed as early as 3-6 hr after treatment, coincided with the selective loss of G0 cells in these cultures, while no significant changes in the proportion of S or G2+M cells were apparent. Agarose gel electrophoresis of DNA isolated from the treated cells indicated degradation of the internucleosomal spacer sections, typical of the endonucleolytic activity which accompanies apoptotic cell death. The data indicate that G0 thymocytes were particularly sensitive to agents that induce apoptosis while cells progressing through the cell cycle were resistant. This suggests that under in vivo conditions (immunological response), the selective death of G0 cells may promote the clonal expansion of stimulated thymocytes which enter the cell cycle. Together with our earlier studies on the effects of CAM and TN on MOLT-4 and HL-60 leukemic cell lines, these data indicate that both, phenotypic- and and cell cycle phase specific- factors modify the ability of cells to respond to toxic agents, including chemotherapeutics by apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS)

From June 1988 to January 1990, 28 patients with primary brain tumours were operated and treated with radiotherapy (RT) (50 Gy whole brain + 10 Gy boost to tumour bed) + cyclohexylnitrosourea (CCNU) 130 mg/msq p.o. every 6 weeks + the radiosensitizer Lonidamine (LND) (150 mg T.I.D. for the whole duration of treatment). Myelotoxicity of this regimen was acceptable, with two cases of grade IV leukopenia and thrombocytopenia requiring discontinuation of treatment. LND was discontinued in 6 patients for major toxicity (myalgias and/or testicular pain), and 3 additional patients required dose reduction of this drug. The median follow-up time of the patients on study was 12 months. The median survival time (MST) was 5 months for grade IV astrocytomas (n = 8) and 16 months for grade III lesions (= 20). No correlation was seen between survival of patients and DNA content, measured by flow cytometry, or levels of O6- alkylguanine-DNA alkyltransferase, an enzyme that repairs the CCNU-induced DNA damage.

The authors investigated the relationship between flow cytometric DNA index (DI, defined as the ratio of the DNA content of malignant cells to that of normal cells) and other prognostic factors (grade and stage, anatomical site, age and sex) with the survival of 115 patients with colorectal cancer. Multiple biopsy specimens from 62 patients were taken during colonoscopy before surgery. Additional samples from 53 patients were obtained from paraffin-embedded material. All patients were treated with surgery only. Fresh-frozen material gave higher incidence of DNA aneuploidy than paraffin-embedded material (79% versus 41%). The patients with DNA diploid tumors (DI = 1) had a better overall survival than those with DNA aneuploid tumors (DI = 1). Among DNA aneuploid tumors, those with DI greater than 1.2 (excluding DI = 2) were worse than those with DI = 1.2 (excluding DI = 1) and DI = 2. Cox's regression analysis showed that pathologic stage was more important for prognosis than DNA index, whereas age, sex, histologic grade, and anatomic site were removed from the analysis as not relevant for prognosis. Relative risk of death (RR), in reference to patients with DI = 1 and Stages A + B (RR = 1), were RR = 1.8 for patients with carcinomas with Stage C. RR = 2.7 for patients with carcinomas with DNA near-diploid and DNA tetraploid tumors. RR = 3.5 for those with DI greater than 1.2 (excluding DI = 2), and RR = 8.0 for those with Stage D. These data indicate that flow cytometrically evaluated DI values have a relevant independent power for predicting the clinical outcome of colorectal cancer patients.

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.

Automated image analyses were performed using Feulgen stained smears of WI-38 cells that were either confluent, or that had received a nutritional stimulus to proliferate 3 hr before collection. These experiments show that it is possible to observe changes in morphometric and densitometric parameters of nuclei that correlate with structural and functional differences in isolated chromatins from quiescent GO and proliferating Gl cells that have been demonstrated by other means. Scatter plot analyses of the data indicated the presence of nuclear images from the stimulated Gl population that had the same deoxyribonucleic acid content as the confluent GO cells, but had greater areas, perimeters and horizontal projections and smaller mean free paths, form factors, and average optical densities. Multiparameter cluster analysis permits, even minimally, an objective, model-independent identification of GO from Gl cells that present an increased nuclear dispersion ( i.e., lower average optical density) systematically accompanied by increased nuclear convolution ( i.e., lower form factor), both compatible with the reported increase in available binding sites with respect to GO cells. The inclusion ofone or more nonproliferating compartments has received considerable emphasis in the construction of recent cell kinetic models. The models are in turn used to estimate the cell cycle parameters of particular populations from physical observables (such as ‘H thymidine incorporation or fluorescence distribution histograms.) Studies of nonproliferating and proliferating cells of the same type have received added impetus as the result of demonstrations of functional and structural differences between chromatins obtained from cells in these states (13, 20). Chromatin structural changes associated with the application of proliferative stimuli to quiescent cells include increases in maximum positive ellipticity in the 250-300 nm region of circular dichroism spectra (3, 14), increased intrinsic viscosity (20), decreased thermal stability (21) and increases in the number of binding sites for intercalating

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.

Numerical experiments were performed to find optimum feature extraction procedures for the classification of mouse L-fibroblasts into Gl, S and G2 subpopulations. From images of these cells different feature sets such as geometric, densitometric, textural and chromatin features were derived which served as data base for the numerical experiments. Linear and nonlinear supervised stepwise learning techniques for the discrimination of the cells into Gl, S and G2 were performed. The classification error was used as criterion for the evaluation of the different numerical feature selection methods. Optimum results were obtained by combining distance based feature selection methods with nonlinear discriminant analysis. The successive solution of 2-class problems improves the results compared to the solution of the 3-class problem. Linear discriminant analysis then may surpass quadratic discriminant analysis.© (1982) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

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.