Pathological and molecular characteristics distinguishing contralateral metastatic from new primary breast cancer.

BACKGROUND: Breast cancer patients have a cumulative lifetime risk of 2%-15% of developing a contralateral metastatic or ex novo primary cancer. From prognostic and therapeutic viewpoints, it is important to differentiate metastatic from second primary. To distinguish these entities, we investigated whether the pattern of X chromosome inactivation could determine whether the two tumors derived from different progenitor cells. MATERIALS AND METHODS: The clonality of bilateral breast cancer was evaluated through the X-inactivation analysis using the human androgen receptor gene (HUMARA) polymorphism and the histopathologic and molecular results were compared. A different or an identical pattern of X inactivation was considered as indicator of a second primary cancer or not informative, respectively. We considered morphological indicators of a new primary cancer the absence of concordance in the histological type or a better histological differentiation. RESULTS: Ten patients with bilateral breast cancer were evaluated. Morphological criteria indicated that eight were second primary, a conclusion confirmed by the X-inactivation analysis. Two cases classified as recurrence according to morphological criteria were classified as second tumor by molecular analysis. CONCLUSION: Our results show that the HUMARA clonality assay can improve the histological parameters in differentiating metastatic cancer from second primary cancer.

Structural differences in the cell binding region of human fibronectin molecules isolated from cultured normal and tumor-derived human cells.

Fibronectins isolated from human plasma (pFN) and from the conditioned media of normal (N-cFN) and tumor (T-cFN) human cells were compared by cathepsin D digestion followed by immunostaining of released fragments with the monoclonal antibody 3E3, specific for the cell binding site. Two different staining patterns were obtained, one specific for pFN and N-cFN, the second common to fibronectins from the 3 different kinds of tumors studied. This indicates structural differences between N-cFN and T-cFN in the cell binding region of the fibronectin molecule.

Interleukin-3 dependent c-myc protein expression during the cell cycle of murine mast cells.

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.

Neurite outgrowth and cell cycle kinetic changes induced by cis-diamminedichloroplatinum II and retinoic acid in a human neuroblastoma cell line.

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.

Cell cycle variations in azoxymethane-induced rat colorectal carcinogenesis studied by flow cytometry.

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.

DNA flow cytometry of endoscopically examined colorectal adenocarcinomas.

The purpose of the study was to evaluate the correlation of DNA-ploidy of colorectal adenocarcinomas (adk) with histological and clinical parameters including the survival of the patients. Multiple biopsies from 95 adk were taken during colonoscopy prior to surgery. The samples were used to obtain nuclei suspensions for specific staining of DNA content and high resolution flow cytometry. DNA-aneuploidy, i.e. the presence of more than one G0/G1 peak, was detected in 67/95 cases (71%). The individual-specific control mucosa was DNA-diploid in all cases. The mean fraction of S-phase cells was 7.2% in control mucosa and 13.6% in adk. DNA-ploidy did neither correlate with Dukes' stage nor with differentiation degree. Among the patients studied for the correlation of DNA ploidy with survival for a period extending to 30 months (n = 51), the DNA aneuploid group was estimated to be about 5 times as risky as the DNA diploid group with respect to the odds of dying. We conclude that DNA flow cytometry of colorectal adk may predict clinical outcome and be helpful in addition to histopathology.

Cell cycle synchronization induced by tamoxifen and 17 beta-estradiol on MCF-7 cells using flow cytometry and a monoclonal antibody against bromodeoxyuridine.

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.

Neuroblastoma cell apoptosis induced by the synthetic retinoid N-(4-hydroxyphenyl)retinamide.

N-(4-hydroxyphenyl)retinamide (HPR) is a synthetic retinoid with anti-cancer properties and lower toxicity than all-trans retinoic acid (RA). Neuroblastoma cells treated with HPR and observed by fluorescence microscopy showed clear signs of apoptosis, such as chromatin condensation and margination, nuclear fragmentation and the presence of "apoptotic bodies". Moreover, measurements on a cell-by-cell basis by the flow-cytometric DNA-content in situ-terminal-deoxinucleotidyl-transferase(TDT) assay showed that apoptosis induced by HPR was dose- and time-dependent and that the fraction of apoptotic cells increased from approximately 15% at 1.25 microM at 2 days after treatment up to approximately 90% at 5 microM and 8 days of continuous treatment. Additionally, we found that cells were induced into apoptosis independently from the cell-cycle phase. In contrast, equimolar or higher doses of RA, from 5 microM to 80 microM, were able to inhibit growth by differentiation, but failed to induce apoptosis. We conclude that the functional effects of HPR and RA in LA-N-5 neuroblastoma cells are mediated by apoptosis and differentiation respectively, suggesting a potential clinical use of HPR in the management of neuroblastoma patients.

DNA flow cytometry of endoscopically examined colorectal adenomas and adenocarcinomas.

DNA ploidy of 64 colorectal adenomas and 49 adenocarcinomas, examined endoscopically, was studied by flow cytometry. We found DNA aneuploidy in none of the 105 normal mucosa samples (0%), in 20 adenomas (31%), and in 36 adenocarcinomas (74%). DNA ploidy of adenomas correlated with size (P = 0.02) and degree of dysplasia (P less than 0.01) but not with histologic type. Adenomas had a 45% incidence of DNA aneuploid stem lines in the DNA index range of 0.80-1.20, compared with 8% in the case of adenocarcinomas. The distribution of the DNA index values of adenocarcinomas was approximately normal, with a mean value 1.63 +/- 0.28. The mean DNA index for the three cases of "carcinoma in adenoma" with invasion of the stalk of the adenoma was 1.52 +/- 0.18. These results, using DNA flow cytometry, provide evidence for the progression of colorectal adenoma to adenocarcinoma. The classification of adenomas according to DNA ploidy may be information of considerable practical value to the clinician in predicting risk of further adenomas and/or risk of cancer.

Flow cytometric DNA ploidy in colorectal adenomas and family history of colorectal cancer.

Flow cytometric DNA ploidy of colorectal adenomas resected from 34 patients and the corresponding patient family history in first-degree relatives were evaluated. The samples with at least two separate G0-G1 peaks were defined as DNA aneuploid. The correlation between DNA ploidy and family history was evaluated using two-by-two contingency tables. This correlation was highly statistically significant: seven of nine patients (78%) with positive family histories, and five of 25 (20%) with negative family history had adenomas with DNA aneuploid stemlines (P = 0.0068). The overall DNA aneuploidy incidence was 12 in 34 cases (35.2%). The combined information of DNA aneuploidy and positive family history of colorectal cancer in patients with colorectal adenomas may help to better understand the process of colon carcinogenesis and to identify patients who have a higher risk for developing a malignancy.

Quantitative analysis of mitotic and early-G1 cells using monoclonal antibodies against the AF-2 protein.

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.

Measurement of c-myc protein content and cell cycle kinetics of normal and spontaneously transformed murine mastocytes by bivariate flow cytometry.

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.

A new method to discriminate G1, S, G2, M, and G1 postmitotic 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 dependent alterations of chromatin structure in situ as revealed by the accessibility of the nuclear protein AF-2 to monoclonal antibodies.

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.

Deletions at chromosome 1p by fluorescence in situ hybridization are an early event in human colorectal tumorigenesis.

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.

Intratumor distribution of 1p deletions in human colorectal adenocarcinoma is commonly homogeneous: indirect evidence of early involvement in colorectal tumorigenesis.

BACKGROUND: Cytogenetics and molecular biology studies have indicated that a large subset of human colorectal adenocarcinomas have distal 1p chromosome arm deletions. The aim of this study was to evaluate the intratumor distribution of 1p deletions under the assumption that homogeneity is an indication of early occurrence. METHODS: Seventy-nine histologically selected primary sectors (40 superficial and 39 deep) and 3 lymph node metastases obtained from 20 human sporadic adenocarcinomas were analyzed. Interphase two-color fluorescence in situ hybridization (FISH) was applied to cytocentrifuged nuclei using a centromeric probe for chromosome 1 and a telomeric probe mapping to the 1p36 band. RESULTS: Deletions at 1p were observed in 35 of 82 tumor samples corresponding to 9 of 20 adenocarcinomas analyzed (45%). Seven of the 9 adenocarcinomas with 1p deletions showed an intratumor presence of these aberrations in all the different tumor sectors. CONCLUSIONS: These data, acquired by FISH interphase cytogenetics, confirm that 1p deletions in colorectal adenocarcinoma are common and suggest that this structural chromosomal aberration occurs mainly as an early event in colorectal tumorigenesis.

Preliminary characterization of a monoclonal antibody (AS-2) against cell cycle related proteins.

A monoclonal antibody (AS-2) raised by using isolated nuclei from a human erythroleukemia cell line as immunogen is described. AS-2 was of IgM type and recognized proteins present in both isolated cytoplasms and nuclei. The molecular weight of the AS-2 recognized proteins in the cytoplasm was 200 kDa and 70 and 60 kDa in the nucleus. The relative amount of these proteins were measured simultaneously with DNA content by flow cytometry. We found the highest protein content (or stainability) for both cells and nuclei in late-G1, S and G2, at approximately the same level, and the lowest content in M and early-G1. Sorting based on DNA content and AS-2 associated fluorescence helped identifying the staining pattern of cells and nuclei. Interphase isolated nuclei and cell cytoplasms were characterized by interdispersed staining over the entire surfaces while mitoses showed two dots only. The present preliminary data indicate that the proteins recognized by the AS-2 monoclonal are cell cycle related and suggest that in mitoses they are associated with the centrosomes.

Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements.

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.