Diploid DNA Values Research Articles

Pulmonary alveolar proteinosis in rats after administration of quartz: its possible role in morphogenesis of lung cancer.

In rats, primary peripheral lung tumors composed predominantly of alveolar type II cells have been induced by inhalation of alpha quartz. In our retrospective study on proliferation markers we evaluated lung specimens of 140 Wistar rats from larger experiments, which had been exposed to Dörentrup quartz (DQ12) by inhalation (10 mg/m3, 56 Weeks, 5 days/week, 7 h/day: n = 27) or intratracheal instillations (5 mg: n = 38; 20 mg: n = 10; 50 mg: n = 28; 15 x 3 mg: n = 12). In the last group 8/12 animals developed lung tumors. Animals were sacrificed 1-32 months after administration. For identification of an increased proliferation of alveolar type II cells the DNA content was monitored by microscopic (static) cytophotometry in histological slides. The argyrophil (AgNOR) method for the demonstration of nucleolar organizer regions (NOR) was used as second marker of type II cell proliferation. Measurements made 24 months after inhalation of DQ12 showed a slight increase of pneumocytic proliferation with 1.64 +/- 0.14 AgNOR/nucleus compared to the controls (1.23 +/- 0.04 mean AgNOR/nucleus). After intratracheal instillation of DQ12 a significant increase of AgNOR was found, e.g. 5 mg: 1.93 +/- 0.23 AgNOR/nucleus (6 months) and 1.96 +/- 0.19 (12 months); 50 mg: 1.77 +/- 15 (6 months) and 2.18 +/- 0.05 (12 months); 15 x 3 mg (+2 ml 2% polyvinylpyridine N-oxide s.c.): 1.81 +/- 0.13 AgNOR/nucleus (27-32 months). With the aid of the 2 c deviation index, i.e. the mean square deviation from the diploid DNA value, it was possible also to identify the pathologically increased proliferation of type II cells after intratracheal instillation of quartz: 0.02 +/- 0.01-0.06 +/- 0.04 c2 (controls); 0.07 +/- 0.04 c2 (5 mg/12 months); 0.12 +/- 0.08 c2 (15 x 3 mg/>27 months) and 0.68 +/- 0.48 c2 (50 mg/12 months). Only in the last group were nearly triploid values detected. Summarizing our results, intratracheal instillation and inhalation of quartz in rats regularly induces alveolar proteinosis and interstitial fibrosis in combination with a dose- and time-dependent increase of the type II cell proliferation rate. As mitogenesis increases carcinogenesis, alveolar proteinosis with increased pneumocytic proliferative activity might be a prerequisite for enhanced tumor development.

Cytofluorometric analysis of spermatocytic seminoma.

Whether spermatocytic seminoma (SS) develops during meiosis or not is still controversial. To determine that, the DNA content of large, small, and intermediate cells of SS was measured by cytofluorometry to obtain DNA histograms for each cell type. The large cells were aneuploid and showed cell cycles with G0/G1 DNA values from 3C to 18C. The ploidy of the large cells doubled stepwise (i.e., 4.5C-9C-18C, 3C-6C-12C, and so forth). The small cells showed distinct cell cycles with a DNA content of 2C or near 2C, and the intermediate cells showed both diploid and aneuploid DNA values. It is speculated that none of the cell types is involved in meiosis, based on the fact that each size of cell appears to have its own cell division cycle. It is concluded that in SS, the diploid cell cycle formation by small cells and the creation of large cells by continuous sequential polyploidization result in the morphologic expression of three types of cells (large, small, intermediate).

Supportive role of image analysis and DNA ploidy pattern in the diagnosis of thyroid tumors.

To evaluate the supportive role of image cytometry and DNA ploidy pattern in the diagnosis of thyroid tumors, a preliminary study was performed on fine needle aspirates of 30 cases. Of these, 10 cases each were of colloid goiter, follicular neoplasm, and papillary carcinoma. The nuclear area and DNA value of 50 cells in each case were measured. The mean nuclear area in colloid goiter (69.50 + 12.62 sq.microns) was significantly lower than the mean nuclear area in a follicular neoplasm (88.71 + 15.51 sq.microns) (P less than 0.05). Similar differences between the mean nuclear area in colloid goiter and papillary carcinoma (124.0 + 12.27 sq microns) was also highly significant (P less than 0.001). The results obtained by image cytometry were compared with estimated DNA ploidy pattern of follicular cells from the same cases. All colloid goiter had mean nuclear area below 100 sq.microns with diploid DNA value. However, papillary carcinoma showed aneuploid DNA pattern in eight cases (80.0%), but mean nuclear area was above 100 sq.microns. A diagnostically useful finding obtained in two of the 10 cases of follicular neoplasm was the association of aneuploid DNA pattern with mean nuclear area of the follicular cells above 100 sq.microns indicating a high probability of carcinoma and thus demanding an urgent open biopsy. These cases were readily distinguished from other cases of the same category showing diploid DNA pattern and mean nuclear area of follicular cells below 100 sq.microns.

Triploidy syndrome in a liveborn female

We present the autopsy report of a liveborn triploid female, born after 36 weeks of gestation, who died at the age of 20 hours. External features were diagnostic: fetal hypoplasia, hypertelorism, microstomia, micro-and retrognathia, preauricular skin tag, low-set ears, and 3-4 syndactylia. All internal organs were hypoplastic. There were atrial and ventricular septal defects. Adrenals and kidneys were fused, the gallbladder was absent, and ovarian hilum cell were found to be hyperplastic. Triploidy, 69xxx, was confirmed cytogenetically. The placenta was hypoplastic and, microscopically, revealed a peculiar type of immaturity, so-called hydatidiform villous hypoplasia, findings which have not been previously reported. We suggest that the generalized fetal and placental hypoplasia and the severe hypoplasia of all internal organs are caused by a proliferative deficiency of the triploid cells. In addition, the nuclear DNA content was determined by cytophotometrically from placental stromal cells and was found to be about 50% above the normal diploid DNA value; i.e., a triploid DNA value was confirmed.

Diploid predominance in hereditary nonpolyposis colorectal carcinoma evaluated by flow cytometry

Fifty-nine colorectal carcinomas of patients with verified cancer family syndrome (CFS) were analyzed for DNA ploidy using flow cytometry. Sixty-eight percent of the tumors were diploid, and 32% were aneuploid. The aneuploid tumors had a median DNA index of 1.24 (range, 1.12-1.97). In 90% of all tumors the DNA index was less than 1.27. This predominance of diploid/near-diploid tumors was seen both in primary and in metachronous carcinomas. In 21 cases a cell cycle analysis was possible. Tumors with the S-phase fraction (SPF) greater than or equal to 9.8% had a worse prognosis than tumors with the SPF of less than 9.8%. These findings suggest that the predominance of diploid/near diploid DNA values is one of the characteristics of colorectal carcinomas in CFS. This might signify the existence of two or more pathogenetically different subgroups of colorectal carcinoma and explain the proposed better prognosis of colorectal carcinoma in CFS compared with other colorectal carcinomas.

Nuclear Dna Recordings in Gastric Carcinoids: A cytofluorometric study on single tumour cells

Cytofluorometric nuclear DNA analyses were performed on single tumour cells from eight argyrophilic gastric carcinoids. Three tumours displayed grossly diploid DNA values, with a fraction of S-phase cells of 1-8%. Four other tumours were also mainly diploid, but showed an increased number of tetraploid cells, and one tumour was aneuploid with a stem-cell line of triploid cells. These and previous results indicate that gastric carcinoids are relatively heterogenous with respect to their morphology, nuclear DNA profiles and biological behaviour, in contrast to most intestinal "classical" carcinoids. Furthermore, the results are in accordance with the suggestion that gastric carcinoids may represent a highly differentiated variety within a spectrum of neuroendocrine neoplasia analogous to the corresponding variety observed in the bronchus (carcinoid-small cell, undifferentiated carcinoma).

Interferon-Induced Nuclear DNA Alterations in Malignant Carcinoid Tumors In Vivo<xref ref-type="fn" rid="FN2">2</xref>

Tumor cell specimens were obtained by ultrasonically guided percutaneous needle liver biopsy from 23 patients with metastatic small intestinal carcinoid tumors. The patients were admitted to the hospital for antitumor therapy (streptozocin, 5-fluorouracil, and leukocyte interferon). The tumor cell samples were used for DNA cytofluorometry. All tumors (12 cases) from untreated patients exhibited diploid DNA stem lines with low proliferative activity. The mean number of tetraploid cells was 8%. Altered nuclear DNA records were obtained in tumors of 8 patients, all of which were treated with leukocyte interferon at 3-6 X 10(6) IU/day for 4-32 months. The DNA records in these tumors varied, but they were mainly in the range between diploid and tetraploid values. In some cases several nuclear DNA peaks were observed within the limits for the cell cycle. It is assumed that the interferon treatment was responsible for the development of altered DNA cell lines because: 1) all untreated tumors yielded regular diploid DNA histograms, 2) interferon was the only drug administered to all patients with tumors displaying altered DNA records, and 3) modal diploid DNA values were observed prior to the interferon treatment in some patients. It is suggested that interferon in vivo blocked carcinoid tumor cell populations in different phases of the cell cycle. This result might explain the nuclear DNA profiles registered in the treated patients as well as the therapeutic effect obtained by interferon.

Nuclear DNA in intestinal carcinoid tumors. A study before and after cytotoxin (streptozotocin and 5-fluorouracil) treatment.

Imprint cytology specimens of metastases of intestinal carcinoids obtained by percutaneous biopsy were analysed cytofluorometrically with regard to nuclear DNA records. All untreated tumors (nine cases) exhibited diploid DNA values with a relatively low proliferative activity (less than 2% nuclei in S-phase region). The mean number of tetraploid cells was 5%. Cytofluorometry also was performed on five tumor metastases treated with the cytotoxin streptozotocin and 5-fluorouracil. After treatment, an increase in the number of tetraploid cells (mean value, 30%) was noted, indicating that the cytotoxin treatment (possibly streptozotocin) on the tumor cells in vivo blocked progression from G2 to M phase. The current cytofluorometric analyses show that diploid nuclear DNA records and a low proliferative activity is a characteristic of malignant carcinoid tumors of the intestine. Due to regular DNA histograms in the carcinoid tumors, it is suggested that reliable studies are permitted of the effect of cytotoxins on the different phases of the cell cycle in vivo.

Nuclear DNA and endocrine activity in carcinomas of the breast.

In a running study of nuclear DNA profiles of female breast carcinomas, 60 consecutive tumors were collected and stained with the Grimelius argyrophil reaction for the identification of endocrine tumor cells. Four (6.7%) tumors were argyrophilic, and they all displayed diploid nuclear DNA values. The argyrophil tumors constituted 20% of the diploid breast carcinomas. One of the tumors was examined electron microscopically and exhibited an abundance of intracytoplasmatic granules of neurohormonal type. In none of the four carcinomas could the endocrine activity be inferred from the morphologic appearance alone, since all could be grouped in regular histopathologic terms. Since the frequency of endocrine cells in unselected materials of breast adenocarcinomas is very low, a relationship exists between diploid nuclear DNA values and with neurohormonal production in these tumors.

Nuclear DNA cytophotometry in prostate carcinoma.

Eighty patients with prostate carcinoma underwent fine-needle aspiration biopsy for cytologic grading and DNA-single-cell fluorescence photometry before and at 6-month intervals after endocrine treatment. The histograms of DNA values showed single peaks and bimodal and scattered distributions which correlated to the different tumor grades before therapy. The DNA values were significantly different from the controls with benign prostatic hypertrophy. After start of therapy, regressive changes of the DNA-histograms were increases of diploid and hypodiploid DNA values and disappearance of secondary peaks. Progressive changes were increased scattering of DNA values and appearance of secondary peaks. Progressive changes in the histograms were closely related to clinical remission and stable disease, but related poorly to clinical progression. The survival correlated with the pretherapeutic DNA-histograms and with the DNA-median, third-quartile, and maximum parameters.

The fluorophenylalanine sensitive and resistant tobacco cell lines, TX1 and TX 4 1. DNA contents, chromosome numbers, nuclear ultrastructures, and effects of spermidine

The two tobacco cell lines TX 1 (wild type) and TX 4 (Palmer andWidholm 1975), which are respectively sensitive and resistant to pfluorophenylalanine, were studied by light and electron microscopy and scanning cytophotometry. The two cell lines differ in the following respects: TX 1 cells exhibit spherical cells, a chromatin organization like in free-grown plants ofNicotiana tabacum, and mainly diploid and haploid DNA values. TX 4 cells show large elongated (thread-like) cells, a diffuse nuclear ultrastructure, and mainly tetraploid and octoploid DNA values. Chromosome number counts indicate that dividing TX 1 cells own a karyotype close to the diploid one, while dividing TX 4 cells are around the tetraploid state. In addition, the effects of an exogenous polyamine, spermidine, on chromatin organization were investigated. While the wild type cell line, TX1, did not respond to the treatment, the mutant, fluorophenylalanine resistant cell line, TX4, exhibited reduced nuclear size and increased chromatin condensation.

DNA contents in Wilms' tumors. A cytofluorometric study.

Cytofluorometric measurement of nuclear DNA was performed on individual tumor cells isolated from paraffin sections of Wilms' tumors. The DNA distribution of one untreated primary tumor showed the first major peak near the diploid range and the second peak in the tetraploid range. There were many cells having amounts of DNA interspersed between the diploid and tetraploid range. Polyploid cells were not observed. Most of the Wilms' tumor cells had a higher diploid DNA value than the small lymphocytes of the control cells. The primary tumor and its metastases showed similar DNA distribution patterns. After treatment, the distribution pattern showed a reduction in number of cells between the diploid and tetraploid range and in the tetraploid range, with the greater number of cells being found in the diploid range. Though the phases of the cell cycle are not always clearly detectable by cytofluorometry, the cells of the untreated Wilms' tumor were distributed into the phases of the cell cycle as follows: 65.9% in G0 and G1 phases; 31.2% in S-phase; and 2.9% in G2 and M phases.

DNS-Ploidie und Proliferationsverhalten beim metastasierten Neuroblastom*

Nuclear DNA contents of tumor cells were measures by fluorescence cytophotometry in bone marrow and touch preparations of 26 children with neuroblastoma, registered in the NBL79 study of the German Pediatric Oncology society. In all cases tumor cells were found to have a higher DNA-content as compared to the normal diploid (2c) DNA-value of granulocytes in the same preparation. Average ploidy values of the histograms of bone marrow metastases were in the tetra- to hypertetraploid (4c-5c) range and showed unimodal distribution in the majority of cases. In the primary tumors there was also a wide distribution of DNA-values in the aneuploid range. A bimodal distribution with stemlines at 2,5c and 5c was found in a stage I, grade I, differentiated primary tumor. Various ploidy patterns found at different sites in the large tumor mass of a newborn might be interpreted as an expression of different degrees of differentiation within the same tumor. In 2 of 4 patients where primary tumor and metastatic lesions could be examined there was a higher ploidy in the 5c to 6c, or even in the 8c range in the metastatic cell population. It appears, thus, that clonal evolution of tumor cells occurs during growth and spread of neuroblastoma.

Are CNS neurons polyploid? A critical analysis based upon cytophotometric study of the DNA content of cerebellar and olfactory bulbar neurons of the bat

A cytophotometric study of the nuclear DNA content of bat cerebellar and olfactory bulbar neurons was performed with particular attention to corrections for distributional error and non-specific light loss and to selection of appropriate control nuclei for the establishment of reliable haploid and diploid DNA values. Feulgen stained sections were measured with an integrating, scanning microdensitometer for correction of distributional error. The values thus obtained were further corrected in 3 different ways to subtract the contribution of background absorbance at 546 nm. Bat haploid (lc) DNA values were derived from spermatozoa, and diploid (2c) values from pancreatic acinar cells, hepatocytes and non-hepatocyte liver cells. Microglia of the olfactory bulb also had 2c values. After correction, all neurons measured, except Purkinje cells, had DNA values more closely approximating the tetraploid (4c) than the diploid (2c) level. Purkinje neurons had mean DNA values closer to 2c than to 4c, but inherent technical difficulties in measuring these very large, light staining nuclei has created less confidence in the values obtained than for those of the other neurons. This uncertainty, plus the apparent existence of two populations of Purkinje neurons, one 2c and the other hyperdiploid, suggested by the DNA distribution curves, make it difficult to eliminate the possibility of polyploidization of Purkinje neurons. A critical analysis of various cytophotometric, radioautographic and biochemical approaches to the problem of CNS neuron polyploidization has revealed potentially serious flaws in many of them, rendering virtually impossible interpretation of the numerous contradictory results in the literature. Standardization of the cytophotometric technique and improvement of the radioautographic approach seem to be prerequisite to the resolution of the existing dilemma.

Flow cytometric DNA analysis of anal canal epithelium and ano-rectal tumours.

Flow cytometric DNA analysis was performed on mucosal biopsies from the various epithelial types normally present in the anal canal, i.e. colo-rectal epithelium, ATZ-epithelium and squamous epithelium as well as from rectal and anal adenocarcinomas and squamous carcinomas of the anal canal. Normal colo-rectal mucosa show homogeneous cell populations with diploid DNA values, while adenocarcinomas contain diploid as well as aneuploid cell populations, regardless of location in the rectum or in the anal canal. The ATZ-epithelium shows a dominating normoploid population with a small "hyperdiploid" peak. Polyploid nuclei were not found. The findings indicate that the ATZ-epithelium is related to metaplastic epithelium rather than to urothelium. The variants of squamous carcinoma in this study were rather poorly differentiated, but the FCM-analysis showed a near diploid population with a high proliferative activity. The number of cases, however, is too small to be conclusive.

DNA and its synthesis in individual tumor cells from human upper respiratory tract squamous cell carcinomas.

Measurements of cytophotometric Feulgen DNA content and incorporation of 3H-thymidine were made in individual tumor cells from 58 patients with squamous cell carcinomas of the head-neck region. These measurements were correlated with a point ranking system for pathological grading for these types of tumors. Two of the parameters of the grading system, mode of invasion and nuclear polymorphism which had been found to be important factors in prognosis in clinical studies were correlated to the objective cellular DNA measurements. In 40 carcinomas, 10 were found to have a predominantly diploid distribution of DNA values, while the remaining 30 tumor values were either multiples of diploid or were aneuploid. Generally, tumors with low point rankings for individual morphological parameters tended to have diploid DNA values. Those tumors with higher DNA values usually had larger point rankings and more advanced clinical stage. When tumor cells from biopsies of 39 patients were studied by 3H-thymidine incorporation, no relevant connections between the labeling index and cellular DNA amounts, malignancy grading, or clinical stage were found.

Identification of young megakaryocytes by immunofluorescence and cytophotometry

The DNA-content of fluoresceine-labeled platelet antigen containing cells of mouse bone marrow was measured. For immunofluorescence highly specific anti-mouse-platelet-serum and fluoresceine-conjugated antigammaglobuline was used, applying the "sandwich" technique. Three hundred panoptically identifable megakaryocytes served as control group. The DNA-polyploidization pattern of megakaryocytes and immunofluorescence positive cells was almost identical. However, among the immunofluorescence positive cells a considerable amount of cells showed DNA-values lower than 4c, whereas the megakaryocytes of the Pappenheim stained smears revealed no DNA-values lower than 4c. The percentages of diploid and tetraploid cells, respectively, was 6 and 7% compared with 0 and 1% of panoptically identifiable megakaryoctyes. The results suggest that young megakaryocytic cells with diploid and tetraploid DNA-values can be detected by immunofluorescence technique, indicating that the flow from the uncommited to the committed megakaryocytic precursor cell appears at this early stage of megakaryocyte production.

Flow cytometry in tumours of the brain

Flow cytometry was used for studying the distribution of single cell DNA content in biopsy material from 85 patients with benign and malignant lesions of the brain and spinal cord. In inflammatory lesions and in most benign tumours, cells with diploid DNA values were observed, sometimes with additional smaller amounts of tetraploid DNA. Cells from malignant tumours were characterized by marked hyperploid DNA values. Flow cytometry is found a rapid and valuable method adding important information to the biological nature of tumours of the central nervous system.

DNA in Non Hodgkin-Lymphoma —A Cytophotometric Study

Cytophotometric DNA determinations on 26 cases of non-Hodgkin's lymphoma yielded the following findings: 1. Follicular centrocytic/centroblastic lymphomas (M. Brill-Symmers) and diffuse centrocytic lymphomas (lymphocytic lymphosarcoma) have a diploid DNA stem line. Diploid DNA values are observed in benign tumors, so that the assignment of these lymphomas to the group of "low grade malignancies" appears justified. 2. Lymphoblastic sarcomas show an aneuploid DNA stem line, as do 96% of all malignant tumors. 3. Lymphoid cells, plasma cells, and immunoblasts seen in immunocytomas are aneuploid. Thus these lymphomas must belong to the group of "high-grade malignant lymphomas" as regards their DNA distribution. 4. Immunoblastic sarcomas have aneuploid DNA stem lines (1 case tetraploid), in which both the lymphoid cells and the plasma cells from those immunoblastic sarcomas arising from immunocytomas show atypical DNTA distribution patterns. 5. In two cases of angioimmunoblastic lymphadenopathy, the lymphoid cells, plasma cells, and immunoblasts are aneuploid. They are thus regarded as "high grade malignancy" lymphomas. The results are discussed with respect to clinical course and prognosis. Measurements on a larger series of cases and correlation to clinical data are needed to support these results. Ultrafast DNA measurements made by flow-through cytophotometry can perhaps be helpful in the future for making the decision between a "low" or "high" grade malignant lymphoma.

The nuclear DNA content of lobular neoplasia of the mammary gland.

Twelve cases of lobular neoplasia and three cases of invasive lobular carcinoma of the breast were examined by Feulgen microspectrophotometry. Two different ranges of nuclear DNA values were observed. Eight cases of lobular neoplasia fell within the diploid range and the four remaining cases within the diploid to tetraploid range, while the three cases of invasive lobular carcinoma showed a DNA distribution between the diploid and tetraploid range. Cytophotometrically, lobular neoplasia with diploid DNA values can be considered to represent a precancerous condition, whereas lobular neoplasia with diploid to tetraploid values are regarded as a more advanced stage of an non infiltrating carcinoma still confined to the performed spaces.