A model for random genetic damage directing selection of diploid or aneuploid tumours.

OBJECTIVES: To test whether genetic instability may determine whether tumours become aneuploid or diploid. MATERIALS AND METHODS: We have identified genes needed for cell survival or replication by combining Affymetrix gene expression array data from 12 experimental cell lines with in silico GEO+GNF and expO databases. Specific loss of heterozygosis (LOHs), chromosomal abnormalities (called derivative chromosomes) and numbers of normal homologues were identified by SNP and SKY analyses. Random gene losses were calculated under the assumption that bi-allelic MMR gene inactivation causes a 20-fold increase in rate of gene loss. RESULTS: There were ∼1.23 × 10(4) genes widely dispersed throughout the genome and possibly expressed by all cells for survival or proliferation, many of these genes performed housekeeping functions. Conservation of the genes may explain the complete haploid genomes found for 15 different cell types and derivative chromosomes selectively retained in aneuploid cancer cell lines after LOH formations, and normal homologue losses. Loss of cell survival/replication genes was calculated to be higher in colon stem cells of carriers of MMR gene mutations than carriers of APC gene mutations. CONCLUSION: Random loss of cell survival/replication genes was calculated to be low enough for colon stem cells with APC gene mutations to 'select' LOH and derivative chromosome combinations favouring tumour cell proliferation. However, cell survival/replication gene loss was calculated to be too high for colonic stem cells lacking MMR genes to survive chromosomal instability, explaining why MMR mutations only produce tumours with diploid chromosome cells.

Strain differences in neointimal hyperplasia in the rat.

We performed an initial screen of 11 rat strains by use of a standard balloon injury to the left iliac artery to observe whether genetically determined differences existed in the development of neointimal hyperplasia. Neointimal hyperplasia was assayed 8 weeks after the vascular injury on coded microscopic sections. Statistically significant differences in the percentages of the vascular wall cross-sectional areas composed of intima (percentage intima) secondary to neointimal hyperplasia were noted among the different rat strains (P<0.02), with the Brown-Norway (BN), Dark Agouti, and Milan normotensive strain rats having the highest and the spontaneously hypertensive rats (SHR) having the lowest percentages of intima. In a separate experiment, F1 hybrids of SHRxBN strains and parental BN and SHR underwent the vascular injury, and the parental strains again showed a statistically significant difference from one another in the mean percentage of intima (P<0. 0001). The F1 hybrids showed an average percentage of intima intermediate between those of the parental strains. The average lumen size of the injured BN vessels were significantly smaller than that of the noninjured control vessels (P=0.044), but this significance disappeared when the circular areas of these vessels were calculated without taking neointimal growth into consideration (P=0.649). These results provide the groundwork for a genetic linkage analysis to identify the genes that influence the development of neointimal hyperplasia after vascular injury.

Evidence for a relatively random array of human chromosomes on the mitotic ring.

We used fluorescence in situ hybridization (FISH) to study the positions of human chromosomes on the mitotic rings of cultured human lymphocytes, MRC-5 fibroblasts, and CCD-34Lu fibroblasts. The homologous chromosomes of all three cell types had relatively random positions with respect to each other on the mitotic rings of prometaphase rosettes and anaphase cells. Also, the positions of the X and Y chromosomes, colocalized with the somatic homologues in male cells, were highly variable from one mitotic ring to another. Although random chromosomal positions were found in different pairs of CCD-34Lu and MRC-5 late-anaphases, the separations between the same homologous chromosomes in paired late-anaphase and telophase chromosomal masses were highly correlated. Thus, although some loose spatial associations of chromosomes secondary to interphase positioning may exist on the mitotic rings of some cells, a fixed order of human chromosomes and/or a rigorous separation of homologous chromosomes on the mitotic ring are not necessary for normal mitosis. Furthermore, the relative chromosomal positions on each individual metaphase plate are most likely carried through anaphase into telophase.

DNA content and other factors associated with ten-year survival after resection of pancreatic carcinoma.

BACKGROUND AND OBJECTIVES: The 5-year survival rates after resection of pancreatic carcinoma have recently increased and are predicted by tumor size, DNA content, and lymph node metastases at the time of resection. However, whether the 10-year survival rates have also increased and are similarly predicted by these factors is not known. METHODS: The influence of preoperative imaging tests, alcohol consumption, cigarette smoking, K-ras mutations, anatomic location, details of surgical resection, pathologic findings, and tumor DNA content on survival was tested for 96 patients after a successful resection of a pancreatic carcinoma with 17 patients being followed for more than 5 years. RESULTS: The 5- and 10-year patient survival rates were 18% and 3%, respectively. Univariate and multivariable analyses showed that tumor DNA content, pathologic tumor size, and lymph node metastases were the strongest prognostic indicators for long-term patient survival, although the importance of tumor size may diminish 2 or more years after resection. Surprisingly, the 11 patients with diploid carcinomas > or = 4 cm had an estimated 10-year survival rate of 36%. CONCLUSION: These results show that the 10-year survival rate for pancreatic carcinoma remains very low, although the subset of patients with biologically favorable tumors has a prolonged survival and possible cure after resection.

Differential effects of nitrogen mustard in vivo on the cancer and host cells in MCa-11 tumours.

We analysed the effects of nitrogen mustard (HN2) on the growth, cell cycle distributions, and ratios of tumour cells to host cells for MCa-11 tumours grown in vivo. Treatment of tumour-bearing BALB/c mice with 3 mg/kg of HN2 produced a significant slowing of MCa-11 tumour growth. Seventy-two hours after treatment in vivo with either 3 or 4 mg/kg of HN2, the host cells in the treated tumours showed a significantly decreased G0/G1 peak and an increased G2/M peak (P < 0.01), whereas the cancer cells in the treated tumours showed significant increases in the G0/G1 peak coupled with relatively decreased proportions of S and G2/M tumour cells (P < 0.001). The ratio of the total number of cancer cells to the total number of host cells in the tumours was significantly increased 72 h after HN2 administration (P < 0.01). Thirty-two days after treatment with HN2, the cell cycle distributions of the host and tumour cells in the treatment and control tumours had returned to being identical, but the ratio of the total number of cancer cells to the total number of host cells remained increased in the treated tumours (P < 0.01). These results show that the administration in vivo of HN2 can lead to entirely different cell cycle effects for the host and cancer cells in the same tumour, and that the partial growth arrest of MCa-11 tumours from HN2 treatment may be due in part to the preferential destruction of host cells rather than solely to a direct cytotoxic effect on the cancer cells.

Overexpression of p53 protein in adenocarcinoma of the pancreas.

Mutations in the p53 tumor suppressor gene are frequently identified in human neoplasms. These mutations may be associated with stabilization and, therefore, with overexpression of the p53 protein product as determined by immunohistochemical staining. Using a new antigen retrieval method and a polyclonal antibody to p53 (CM-1), the authors examined 48 formalin-fixed paraffin-embedded adenocarcinomas of the pancreas for overexpression of the p53 gene product. These 48 carcinomas were obtained from a series of patients with well-documented clinical histories and extensive follow-up. The carcinomas had been analyzed previously for K-ras gene mutations, tumor ploidy, and tumor proliferating index. Specific diffuse nuclear staining for the p53 protein was identified in 19 of the 48 (40%) infiltrating carcinomas examined. Focal or negative staining was seen in the remaining 29 cases (60%). In addition, 17 of the neoplasms contained synchronous in situ carcinomas; two (12%) of these displayed diffuse nuclear staining for the p53 protein. Overexpression of p53 was associated with aneuploidy (P = .05), which had been a poor prognosticator in this series of adenocarcinomas of the pancreas. Although overexpression of p53 appeared to be associated with poor prognosis (hazard ratio, 1.8; P = .07), this was not statistically significant. Overexpression of p53 was not significantly associated with K-ras oncogene mutations or tumor proliferating index. The authors conclude that overexpression of the p53 protein occurs frequently in invasive adenocarcinomas of the pancreas and in some in situ carcinomas, as well.

Measurement of DNA content and of tritiated thymidine and bromodeoxyuridine incorporation by the same cells.

We tested a method of measuring DNA content (Feulgen) and tritiated thymidine ([3H]-T) and bromodeoxyuridine (BrdU) incorporation by the same cell. Initial experiments showed that Feulgen hydrolysis denatured the DNA of fixed cells sufficiently to allow detection of incorporated BrdU with monoclonal antibodies. MCa-11 cells were then double-labeled with [3H]-T and BrdU, placed on slides, and Feulgen stained. Next, absorption cytometry was performed to measure the DNA content of randomly selected cells. Feulgen staining and the development and removal of either the [3H]-T or the BrdU grains after DNA measurements did not interfere with subsequent detection of the grains from the other label, and BrdU and [3H]-T can be used reliably in combination for identification of S-phase cells. This method may eventually allow the use of microscope-based image analysis to selectively measure the DNA contents and the BrdU/[3H]-T labeling of non-transformed stromal and cancer cells in solid tumors, thereby providing new insights into the growth kinetics of these heterogeneous cell populations.

Impact of axillary lymph node dissection on the therapy of breast cancer patients.

PURPOSE: We studied a series of 283 breast cancer patients retrospectively to determine the actual benefits of axillary lymph node dissection (ALND) for these patients. PATIENTS AND METHODS: The records of 283 women with invasive breast cancer treated between 1988 and 1990 were reviewed for histologic status of the axillary lymph nodes, tumor size, DNA content, hormone-receptor values, and actual adjuvant treatments received. RESULTS: ALND was of possible therapeutic benefit for the 15% (43 of 283) of patients who had clinically positive nodes. Nodal metastases were found in 86% (37 of 43) of patients in this subgroup. ALND alone determined the indication for standard adjuvant therapy for a group of 31% (88 of 283) of patients who had favorable primary biopsy findings and clinically negative axillary nodes; ALND proved that 13% (11 of 88) of these latter patients had positive nodes. For 54% (152 of 283) of patients who had clinically negative nodes and unfavorable biopsies, ALND played no role in the decision as to whether standard adjuvant therapy was indicated. Only 5% (seven of 152) and 3% (four of 152) of these latter patients received radiation therapy and/or high-dose adjuvant chemotherapy, respectively, because of ALND. CONCLUSION: The benefits of ALND vary greatly for different groups of breast cancer patients, and controlled studies may be needed to determine whether ALND is necessary for all breast cancer patients.

K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization.

We examined 82 surgically resected or biopsied, formalin-fixed, paraffin-embedded primary adenocarcinomas of the pancreas for the presence of activating point mutations in codon 12 of the K-ras oncogene. Mutations were detected using primer-mediated, mutant-enriched, polymerase chain reaction-restriction fragment length polymorphism analysis and characterized further by allele-specific oligonucleotide hybridization. This combination of mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis and allele-specific oligonucleotide hybridization results in a rapid and sensitive characterization of the mutations in codon 12 of K-ras. Sixty-eight (83%) of the 82 carcinomas examined harbored a point mutation. Of the 68 mutations, 33 (49%) were guanine to adenine transitions, 27 (39%) were guanine to thymine transversions, and eight (12%) were guanine to cytosine transversions. Mutations were found in carcinomas of the head (61 of 75, 81%) as well as in carcinomas of the body or tail (seven of seven, 100%) of the pancreas. The overall prevalence of K-ras point mutations in adenocarcinomas of the pancreas obtained from patients who smoked cigarettes at some point during their lives (88%; 86% in current smokers and 89% in ex-smokers) was greater than that seen in pancreatic adenocarcinomas from patients who never smoked cigarettes (68%, P = 0.046). The presence of K-ras point mutations did not correlate with tumor ploidy, tumor proliferating index, or patient survival. These results demonstrate that primer-mediated, mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis combined with allele-specific oligonucleotide hybridization can be used to detect and characterize mutations in codon 12 of the K-ras oncogene in formalin-fixed, paraffin-embedded tissues, and the results confirm that activating point mutations in codon 12 of the K-ras oncogene occur frequently in adenocarcinomas of the pancreas.

Evidence for altered cell-cycle traverse of the non-modal cells of the heteroploid MCa-11 line.

Classic stem cell theory states that the growth of heteroploid cell populations is due to the proliferation of 'main stemline' cells with modal DNA content and chromosome number. Cells with non-modal DNA content and chromosome number are thought to be blocked and/or destroyed at mitosis. To test this, we studied two chromosomally stable cell populations (mouse bone marrow and WCHE-5 cells) and one heteroploid, chromosomally diverse cell line (MCa-11). The heteroploid MCa-11 cells showed significant [3H]dT labelling for cells with DNA contents below the modal G0/G1 peak and above the modal G2 peaks (P < 0.001). This was consistent with the presence of cells with the non-modal DNA content that were engaged in replicative DNA synthesis. A percentage labelled mitosis analysis showed that MCa-11 cells with non-modal DNA content and chromosome number were able to complete mitosis, although with prolonged pre-karyokinetic time. These results suggest that many non-modal cells present in heteroploid cell populations are capable of continued proliferation.

A comparison of flow cytometric and absorption cytometric DNA values as prognostic indicators for pancreatic carcinoma.

BACKGROUND: The DNA content of 30 adenocarcinomas of the head of the pancreas was measured by flow and absorption cytometric analysis. METHODS: Each of the patients in this study had curative pancreatoduodenectomy. The absorption cytometric measurements were done in a research laboratory, and the flow cytometric measurements were performed in a commercial laboratory. The DNA measurements were done on nuclei disaggregated from pancreatic cancer tissue blocks without the examiner knowing whether the patient had survived. RESULTS: Twenty-one of the 30 cancers were found to be aneuploid by absorption cytometric analysis, whereas only 1 of the 30 cancers was aneuploid by flow cytometric analysis. This difference was statistically significant (P < 0.001). CONCLUSIONS: Univariate and multivariate analyses showed that the absorption cytometric DNA measurements were stronger prognostic determinants for patient survival than were the flow cytometric DNA measurements, indicating that some caution may be warranted in the interpretation of commercially obtained DNA distributions of pancreatic carcinomas.

Non-random distribution of abnormal mitoses in heteroploid cell lines.

We have performed absorption-cytometric DNA measurements of the DNA contents of the G0/G1, G2, metaphase, and telophase cells of the heteroploid MCa-11 and HL-60 lines, as well as the WCHE-5 line which has a narrowly restricted number of chromosomes. We found that morphologically unbalanced mitoses occurred much more frequently in telophase-cell pairs of the heteroploid MCa-11 and HL-60 lines than in those of the chromosomally stable WCHE-5 line. Furthermore, the morphologically unbalanced mitoses represented unequal segregation of DNA into each of the daughter telophase nuclei. Such mitotic segregation errors (MSE) occurred almost exclusively in telophase cells with DNA contents which were above, or below, the DNA content of the modal telophase population. The net effect of these non-random, unblanced divisions of heteroploid cells with non-modal DNA contents is to produce one daughter cell with a DNA content that tends to return to the modal DNA content peak.

Pancreatic cancer cell DNA content correlates with long-term survival after pancreatoduodenectomy.

The DNA content of 47 adenocarcinomas arising in the head of the pancreas from patients who had undergone successful pancreatoduodenectomy was measured. The DNA measurements of each tumor were made without knowledge of the clinical course by absorption cytometry performed on Feulgen-stained nuclei that had been disaggregated from pancreatic cancer tissue blocks. Forty-seven evaluable DNA distributions were obtained from specimens taken between 1975 and 1988. Of the 47 tumors, 19 (40%) were diploid and 28 (60%) were aneuploid cancers. The 19 patients with diploid cancers had a median survival time of 25 months. Median survival of the 28 patients with aneuploid cancers was 10.5 months. This difference was statistically significant (p = 0.003). A multivariate life table regression analysis demonstrated that the ploidy and proliferative index as determined by absorption cytometry were independent prognostic factors, as strong as or stronger than the number of positive nodes and tumor size. Thus cellular DNA content appears to be one of the most important predictors of survival in patients with adenocarcinoma of the head of the pancreas who have successfully undergone a pancreaticoduodenectomy.

Discrepancies among the metaphase, telophase, and the G0/G1 and G2 DNA peaks of heteroploid cell lines.

Heteroploid cell populations often show narrow peaks of G0/G1 and G2/M DNA content and broadly distributed chromosome numbers. This was originally explained by the selective metaphase arrest of the cells that have non-modal chromosome numbers. To test whether this explanation applies, we have measured the chromosome number distributions, as well as the G0/G1, G2, metaphase (M), and telophase (T) DNA distributions, of the cell lines WCHE-5, MCa-11, and HL-60. The WCHE-5 cells had narrowly distributed chromosome numbers and G0/G1 G2, M, and T DNA peaks. The MCa-11 and HL-60 cells also had narrowly distributed G0/G1 and G2 DNA peaks, but broadly distributed chromosome numbers and M and T DNA peaks. The widths of the MCa-11 and HL-60 M- and T-cell DNA peaks were similar to those of their chromosome number peaks, suggesting that all cells were completing mitosis, regardless of chromosome number or DNA content. Thus, selective metaphase arrest does not seem to be the cause of the narrow G0/G1 and G2 DNA peaks of heteroploid cell populations.

An organized system of trauma care in an Army community hospital.

The use of a Trauma Team in a United States Army Medical Activity is described. Feasibility of implementation, logistical considerations, results, and applicability to other Armed Forces Hospitals are discussed.

Development of polyploidization in taxol-resistant human leukemia cells in vitro.

The effects of taxol on antitubulin immunofluorescent staining patterns, cellular DNA content, and labeling with [3H]thymidine were measured for the taxol-sensitive HL60 and taxol-resistant K562 cell lines after exposures for 0, 4, 12, and 24 h. Taxol caused a relative increase in the fraction of 4C interphase and metaphase cells in both lines although the 4C interphase accumulation was greater for the resistant K562 line. Of the cells with S-phase DNA content, taxol-treated HL60 cells were less likely to incorporate [3H]thymidine than taxol-treated K562 cells. However, a decrease in percentage of S-phase labeling for both lines relative to control cells was seen. Finally, taxol induced the development of polyploid cells (cells with DNA contents greater than that of the 4C G2-M peak) in the relatively taxol-resistant K562 cells, an effect not seen in the relatively taxol-sensitive HL60 line. After 24 h of taxol exposure 70% of all K562 cells were polyploid while only 8% of the HL60 cells were polyploid. The capacity of K562 cells to generate polyploidy in response to taxol correlated with taxol resistance by previous assay and may be a useful indicator of drug resistance.

Demonstration of the cell cycle positions of taxol-induced "asters" and "bundles" by sequential measurements of tubulin immunofluorescence, DNA content, and autoradiographic labeling of taxol-sensitive and -resistant cells.

We used reliable and relatively inexpensive equipment to make sequential sets of measurements of antitubulin immunofluorescence, Feulgen staining, and autoradiography on the same cells. This was done to evaluate tubulin conformations, DNA content, and [3H]-thymidine incorporation in cell lines sensitive (HL60) and resistant (K562) to the novel anti-tubulin chemotherapeutic agent taxol. Numbers of cells with microtubule bundles have been found to correlate with sensitivity to taxol by clonogenic assay for several leukemic cell lines. We have found that cells with "asters" produced by taxol exposure are in mitosis and that cells with taxol-induced "bundles" are in G0/G1, S, and G2 phases. We further found that S-phase cells with microtubule bundles in both sensitive (HL60) and resistant (K562) cell lines were able to incorporate [3H]-thymidine after 4-hr exposure to taxol. As microtubule bundles and asters occur in cells of the same cell cycle phases in both lines, we conclude that the greater frequency of cells with microtubule bundles reported for sensitive cells after taxol treatment cannot result from drug exclusion nor from different effects of the drug on cell microtubules in these two leukemic lines.

Acid giemsa technique for rapid identification of mitotic cells.

We developed a rapid technique for differential staining of compacted chromatin as a tool for screening of large tissue culture cell populations for mitotic cells. With a combination of acid Giemsa staining and counterstaining, differential staining of mitotic cells and classification according to stage of mitosis can be accomplished at magnifications as low as x 50-100 (objectives of x 5-10). The mapped and classified cells can then be de-stained and re-studied for DNA content by Feulgen staining and/or for uptake of radioactive DNA precursors by autoradiography. The staining and de-staining procedures outlined do not affect the reproducibility and accuracy of DNA content measurements or measurements of radioactive uptake. Therefore, this technique can be used for cell kinetic analysis by the percentage labeled mitoses method and for cytophotometric studies of mitotic segregation.

Differences in the flow and absorption cytometric DNA distributions of mouse hepatocytes and tumor cells.

We have determined the DNA content, the ploidy levels, and the percentages of different cell types present in small and large mouse mammary tumors as well as in young and old mouse livers by using absorption and flow cytometry. Absorption cytometry data indicated a significant increase in the proportion of transformed G0/G1 cells in the tumors as compared to that of the stromal G0/G1 cells with progressive tumor growth. This increase was not detected by flow cytometry. In both young and old mouse livers, a small number of cells of higher ploidy (8C and 16C) were detected by absorption cytometry but were not apparent in histograms obtained by flow cytometry. Furthermore, changes in the proportions of liver cells of different ploidy with age were apparent in absorption cytometry data but not in flow cytometry data. In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.

Slowing of cell cycle traverse for cells in exponential monolayer cultures placed into plateau-fed and starved medium.

MCa-11 tumor cells in exponential monolayer cultures were pulse/chase-labeled with [3H]thymidine and then regrown in fresh, plateau-fed, or starved medium. We measured the DNA content and autoradiographic labeling of these cells by absorption cytophotometry at intervals of 0, 2, 4, 8, 12, and 24 h to follow the progress through the cell cycle of those cells which had incorporated isotope. We found that for the cells grown in plateau-fed and starved medium the G0/G1, S, and G2 phases of the cell cycle were prolonged when compared to those for cells grown in fresh medium. These results show that, under adverse microenvironmental conditions, the growth of tumor cells can be regulated in all phases of the cell cycle, and that this regulation can include lengthening and even cessation of replicative DNA synthesis.