Aneuploid Cell Lines Research Articles

Extravillus dividing fetal cells at CVS: evidence of their erythroblastic origin.

Cytological and cytogenetic studies were performed on nucleated fetal cells present in chorionic villus transport medium. The erythroblastic origin of these cells was shown. Fetal erythroblasts in spontaneous mitosis were frequently observed; chromosome counts were obtained from them but poor quality often prevented banded analysis. Cytogenetic study of erythroblast metaphases can be useful as an additional diagnostic aid in cases of mosaicism with aneuploid cell lines.

COMPARISON OF FLOW CYTOMETRIC DNA CONTENT IN THE PRIMARY TUMOR AND IN THE CORRESPONDING LYMPH-NODE METASTASIS OF PATIENTS WITH SQUAMOUS CELLS CARCINOMAS OF THE HEAD AND NECK

The clonal DNA content of the cell populations of primary tumors and corresponding lymph node metastases was analysed by DNA flow cytometry in 48 patients with squamous cell carcinoma of the oral cavity. Three primary tumors were diploid, two associated with diploid metastases, one showing an aneuploid cell line in the secondary lesion. Fourty-five cases were aneuploid in both the primary tumors and in the synchronous metastases, all characterized by nearly identical DNA indices of the corresponding primary and secondary lesions. With few exceptions, aneuploidy formation appears to take place prior to dissemination of metastatic cells.

Flow cytometric analysis of cytokinetics of L3-acute lymphoblastic leukemia/lymphoma

Cell kinetic differences have been described between acute lymphoblastic leukemia with L1 and L2 morphology. We now report cytokinetic and DNA ploidy findings of the rare L3 B-cell leukemia/lymphoma. Flow cytometry analysis of nineteen samples was performed by simultaneous DNA-RNA staining with acridine orange. RNA and DNA indices and cell cycle distributions were calculated. The RNA-index of the G 0/G 1 cells was 17.9 ± 8.7 and the number of cells in S phase and S + G 2M were 21 ± 10.6 and 28.0 ± 13.9 percent respectively. DNA aneuploidy was found in 6/19 (31.6%) and in two cases multiple aneuploid cell lines were observed. DNA aneuploidy and multiple abnormal stemlines adversly affected survival ( p < 0.05), while kinetic parameters did not affect survival ( p > 0.05). The cytokinetic data are significantly different (S phase and RNA-I; p < 0.001) than previously reported for the L1 and L2 ALL. Abnormal DNA stemlines were found in cases with no detected cytogenetic abnormalities. This study confirms that L3 ALL is characterized by significantly increased proliferation and provides a means for a flow cytometric identification of this subtype as compared to L1 and L2 ALL.

Establishment and characterization of a mouse Schwann cell line which produces myelin in vivo.

A Schwann cell line (MSC 80) was established from purified mouse Schwann cell cultures using large doses of serum. MSC 80 cell line is an aneuploid cell line which has a doubling time of 17 hr and has been maintained through more than 110 passages. Most of MSC 80 cells are of bipolar or stellate (3-5 processes) shape. A few others are irregular in shape, gigantic, and multinucleated. All MSC 80 cells express antigens of myelin-forming Schwann cells such as S-100, 224/58, laminin, and other glycoproteins of the extracellular matrix. However, they also express the non-myelin-forming Schwann cell antigen GFAP. By time-lapse cinematography, MSC 80 cells exhibit the Schwann cell characteristic rhythmical undulations. When induced to form aggregates in agar, they form intercellular junctions and basement membrane-like structures. In addition, after transplantation in or at a distance from a lysolecithin induced lesion, MSC 80 cells form myelin around the host demyelinated axons. MSC 80 cells thus express, when isolated in vitro, some of the normal myelin-forming Schwann cell phenotype. In addition, they present the major advantage of forming myelin when associated with axons in vivo.

DNA Analysis in Predicting Survival of Irradiated Patients with Transitional Cell Carcinoma of Bladder

A total of 115 patients with invasive transitional cell carcinoma of the bladder underwent radical radiotherapy between 1975 and 1986 and were followed up until the end of 1990. Apart from routine clinical observations, flow cytometric DNA measurements made on fresh tumour material were available for analysis. Actuarial cancer-free survival controlling for response to treatment was analysed with the log-rank test, bivariate and multivariate analyses using Cox's stepwise regression model on probable prognostic factors. The overall actuarial 5-year cancer-free survival rate was 30%. Survival was significantly correlated with response to treatment: 59% for patients with complete regression and 5% for those with residual tumour. Prognostic factors that significantly correlated with death from cancer were advanced stage, large size, incomplete resection, ureteric obstruction, anaemia, carcinoma in situ grade 3 and occurrence of more than one aneuploid cell population. However, only 3 of these factors were of independent power in the multivariate analysis: stage, size and carcinoma in situ. Of 21 patients with a history of primary or secondary carcinoma in situ, 19 died from cancer during follow-up: 18 of the 21 patients had tumours that were aneuploid with more than one aneuploid cell population. It is concluded that curative radiotherapy can be successful only in patients with less advanced tumours assessed according to clinical stage and size, aneuploid tumours with not more than one aneuploid cell line, no carcinoma in situ, no ureteric obstruction, and in whom a complete transurethral resection of the exophytic tumour is possible.

New Developments in the Pathologic Diagnosis of Adrenal Cortical Neoplasms: A Review

In the past decade, our knowledge of neoplasms arising in the adrenal cortex has been expanded greatly. Histologic criteria for distinguishing benign from malignant adrenal cortical neoplasms have been developed. In this review, three systems useful in making this distinction are reviewed and compared. Pathologic indicators of prognosis for adrenal cortical carcinomas have been proposed and these include mitotic rate, stage, surgical resectability, nuclear grade, and tumor size. Of these, mitotic rate appears to be the best indicator. Adrenal cortical carcinomas with a high mitotic rate behave most aggressively. The role of immunohistochemical and DNA content analysis in the diagnosis of adrenal cortical neoplasms is limited. Neoplastic adrenal cortical cells contain a low density of keratins that is often destroyed by routine fixation and paraffin embedding. Thus, the absence of keratins in adrenal cortical neoplasms, particularly carcinomas, in routinely processed tissue should not dissuade the pathologist from making the diagnosis of an epithelial neoplasm. DNA content analysis has revealed an imperfect correlation between DNA ploidy and histologic diagnosis. Some adrenal cortical adenomas contain aneuploid stem cell lines, whereas some adrenal cortical carcinomas have diploid DNA content. Molecular genetic analyses suggest that one or more tumor suppressor genes may be involved in the pathogenesis of adrenal cortical neoplasms.

Clinical Significance of Flow Cytometric Deoxyribonucleic Acid Measurements of Deparaffinized Specimens in Bladder Tumors

A retrospective study of flow cytometric measurements on paraffin-embedded tumor specimens from 188 patients with bladder tumor was conducted. Results were analyzed in combination with the morphological variation in bladder tumors. It was found that the deoxyribonucleic acid (DNA) ploidy pattern, the degree of infiltration and the multiplicity of bladder tumor were closely related with tumor recurrence, among which the DNA ploidy pattern was most significant. In aneuploid bladder tumors, the recurrence rate and the mean annual recurrence frequency were 76.7% and 1.46, respectively, and in the diploid bladder tumors, they were 18.7% and 0.33, respectively. Aneu- ploidy was the most indicative parameter of recurrence in bladder tumors. In addition, according to the DNA content and the ploidy level of the aneuploid cell lines, the aneuploid tumors in our group were divided into four types, namely tetraploid tumors, noneuploid tumors with an DNA index (DI) <1.5, hypotetraploid tumors and aneuploid tumors with several cell lines. The results showed that the recurrence rate of tetraploid tumors was relatively lower, and it became higher and higher in the following order: noneuploid tumors with DI < 1.5, noneuploid tumors with DI > 1.5, and two-aneuploid tumors. This indicates that there are different biological behaviors in tumors with different ploidy patterns. Finally, the correlation between the DNA ploidy pattern and tumor métastasés was also discussed.

Relative order and location of DNA sequences on chromosome 21 linked to familial Alzheimer disease

Recently, a gene causing familial Alzheimer disease (FAD) was linked to DNA probes on chromosome 21 by genetic analysis. To investigate the precise physical location of these DNA probes, we have constructed a physical map of this region of chromosome 21 by using quantitative Southern blot analysis of cell lines aneuploid for parts of chromosome 21. The following DNA sequences were investigated: D21S16, D21S13, FB68L (cDNA probe for the amyloid protein precursor [APP] gene), and D21S1. We find that all DNA probes are located in the same region of chromosome 21, in q11.2-q21.05. We further show that D21S16 must be centromeric to D21S13, because D21S16, but not D21S13 is present in one copy in a cell line with deletion of the region 21pter-21q 11.2. High resolution chromosome analysis is presented to define this breakpoint. This new panel of aneuploid cell lines will allow the rapid mapping of new DNA probes in the vicinity of the FAD gene.

Confirmation of CVS mosaicism in term placentae and high frequency of intrauterine growth retardation association with confined placental mosaicism

About 2 per cent of specimens from chorionic villus sampling (CVS) analysed either on direct preparation of cytotrophoblast cells or after culture of mesenchymal stroma reveal confined placental mosaicism (CPM), most commonly involving chromosomal trisomy. A significantly higher rate of prenatal loss (22 per cent) as well as the presence of intrauterine growth retardation (IUGR) has been reported among pregnancies with CPM. To evaluate more precisely the effect of these aneuploid cell lines confined to the placenta on intrauterine fetal growth and fetal survival, we have studied 34 term placentae from pregnancies with CPM diagnosed on CVS and confirmed identical mosaicism in 17 of these placentae. There was a direct correlation between a high number of aneuploid cells present at CVS and a high likelihood of their detection in term placenta. Also, the proportion of aneuploid cells in the mosaic term placentae correlated with that observed in CVS specimens. Among 17 gestations with confirmed CPM at delivery, there were six cases of IUGR identified, five in liveborns and one associated with intrauterine death.

Toxicity of Pulpispad using four different cell types

The cytotoxic effect of a zinc oxide-eugenol-based paste (Pulpispad) was evaluated in vitro after setting for 1 day and 1 week. Target cells were L929 cells, gingival, periodontal ligament and pulpal fibroblasts. The material was incubated with the cells for 4 and 24 hours, and its toxicity was evaluated with the 51Cr-release method. Pulpispad was highly cytotoxic to all cell lines even after setting for 1 week. The use of Pulpispad is not recommended for future clinical application. The various responses among the four cell lines indicated that diploid cell lines can, under certain circumstances, be less sensitive than aneuploid cell lines. It is therefore suggested that in the evaluation of biomaterials the choice of cell lines should be carefully considered, as they can display varying sensitivities.

Adrenal cortical carcinoma: Flow cytometricstudy of 22 cases, an ECOG study

Flow cytometric studies of archival material from 22 adrenal carcinomas demonstrated aneuploidy in 21 of 22 cases. Heterogeneity of nuclear DNA was found in 14 of the 22 cases. Eight of these showed distinct aneuploid and diploid populations, and 6 showed multiple aneuploid cell lines. The heterogeneity was detected because numerous paraffin-embedded samples of each tumor could be examined. No correlation was found between either aneuploidy or heterogeneous DNA content and patient survival, response to therapy, or hormone secretion.

The clinical significance of flow cytometric deoxyribonucleic acid measurement of deparaffinized specimen in bladder tumor

A retrospective study of flow cytometric measurements on paraffin-embedded tumor specimens from 188 patients with bladder tumor was conducted. Results were analyzed in combination with the morphological variation in bladder tumors. It was found that the deoxyribonucleic acid (DNA) ploidy pattern, the degree of infiltration and the multiplicity of bladder tumor were closely related with tumor recurrence, among which the DNA ploidy pattern was most significant. In aneuploid bladder tumors, the recurrence rate and the mean annual recurrence frequency were 76.7% and 1.46, respectively, and in the diploid bladder tumors, they were 18.7% and 0.33, respectively. Aneuploidy was the most indicative parameter of recurrence in bladder tumors. In addition, according to the DNA content and the ploidy level of the aneuploid cell lines, the aneuploid tumors in our group were divided into four types, namely tetraploid tumors, noneuploid tumors with an DNA index (DI) less than or equal to 1.5, hypotetraploid tumors and aneuploid tumors with several cell lines. The results showed that the recurrence rate of tetraploid tumors was relatively lower, and it became higher and higher in the following order: noneuploid tumors with DI less than or equal to 1.5, noneuploid tumors with DI greater than 1.5, and two-aneuploid tumors. This indicates that there are different biological behaviors in tumors with different ploidy patterns. Finally, the correlation between the DNA ploidy pattern and tumor metastases was also discussed.

DNA-RNA顕微蛍光多重測光法を用いた膀胱腫瘍生物学的悪性度の検討

An acridine orange cytofluorometric analysis was used to study the DNA-RNA contents of bladder washing exfoliated cells from 109 patients with transitional cell carcinoma of the urinary bladder. DNA and RNA measurements were performed in each of 200-300 cells/sample, and DNA index (DI) and RNA index (RI) were calculated from the distributions of these values. Presence of aneuploid stem cell line and/or increased mean DNA contents were observed in 83 (76.1%) of the 109 patients. Elevated RNA content in a diploid cell population, was of no additional diagnostic significance. Histologically diagnosed high grade and high stage bladder carcinoma apparently showed an aneuploid stem cell line with high DI and high RI. Thirty patients with superficial bladder cancer (Ta and Tl) who had tumor recurrence within 2 years after surgical interventions showed a significantly increased RI in contrast to 10 patients who had no recurrence for more than 2 years. RNA content measurement was thought to be useful for prediction of tumor recurrence after surgical interventions. Six patients who had a progression from superficial to invasive carcinoma were with high histological malignancy, lamina propria invasion, high DI and high RI. However, there was no significant difference in RI between the 6 patients and the other patients with superficial bladder cancer. Eight patients who had distant metastasis within one year after radical cystectomy also showed a significant increase in RI in contrast to 8 patients who had no metastasis for more than 1 year. Increased RI may be said to be an important risk factor in predicting the development of distant metastasis and recurrent tumor after surgical interventions.

Precision of DNA flow cytometry in inter‐institutional analyses

A Bladder Cancer Flow Cytometry Network study has been carried out to further identify and quantify sources of inter- and intra-laboratory variability. Replicate samples containing four mixtures of peripheral blood lymphocytes and aneuploid cell lines were distributed together with reference standards to six laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Two of each of the four sample types and a reference standard were analyzed by each laboratory on 3 separate days to obtain cellular DNA distributions. DNA index (DI) and hyperdiploid fraction (HDF) were calculated for each histogram using an automated technique. The results showed significant inter- and intra-laboratory differences. Results were evaluated by a two-way analysis of variance to estimate components of the overall variation attributable to individual sources. Error variation was found to be the major component of random variation. Specimen means were also compared for each laboratory. No significant differences were noted in mean DI for similar specimens; however, agreement in HDF between similar specimens was lacking in most laboratories. Prediction intervals were computed to estimate the range of values expected for a single specimen based on the analysis of the previous six. Prediction intervals for DI were quite good while those for HDF were troublesome due to wide variation. The results of these studies indicate that intra- and inter-laboratory variability are high enough that results for a single sample may not be sufficiently precise to allow comparison to results obtained in other laboratories.(ABSTRACT TRUNCATED AT 250 WORDS)

Modal DNA Values and Estramustine-Binding Protein (EMBP) as Prognostic Markers in Prostatic Cancer

In this study, we have investigated the model DNA values and the expression of estramustine-binding protein (EMBP) in formalin-fixed and paraffin-embedded TUR specimens from 76 untreated patients with prostatic cancer. In addition, specimens from 13 patients were analyzed for tumour EMBP expression only. Ploidy was measured as diploid, tetraploid and non-tetraploid aneuploid or aneuploid in the near-diploid region. All patients had been referred during 1978-1981, and were subjected to TUR due to urinary obstruction. Survival data were obtained for all patients through March 1988. Statistical analyses were performed using a Cox's regression model with respect to survival and cause specific survival and correlated to the DNA pattern and the expression of EMBP. The existence of a near-diploid aneuploid cell population as well as poor differentiation grade were both statistically significantly correlated with poor survival. Near-diploid aneuploid cell lines were seen in 9/76 (12%) of the patients and were also seen in well differentiated cancers (4/17). The expression of EMBP was most abundant in the moderately differentiated cancers. However, all prostatic cancer specimens investigated were positive for the antigen. Patients with poorly differentiated carcinomas and high EMBP expression showed a tendency towards better prognosis than those with poorly differentiated carcinomas and low EMBP expression. The present patient material was, however, too small to show a statistically significant correlation between EMBP and survival.

Deoxyribonucleic Acid Flow Cytometry in Predicting Response to Radical Radiotherapy of Bladder Cancer

Using flow cytometry tumors can be classified according to their deoxyribonucleic acid content as diploid or aneuploid with 1 or several cell lines and to the ploidy level of the aneuploid cell lines, as well as to the proportion of proliferating cells. We used this technique in 73 patients with invasive transitional cell carcinoma of the bladder who received radical radiation therapy with followup of surviving patients for at least 5 years. The effects of therapy were related to the deoxyribonucleic acid patterns before irradiation. All diploid tumors disappeared, while aneuploid tumors with 1 cell line disappeared in 55% and those with several cell lines in 30% of the patients. Aneuploid tumors with cell lines exceeding 4c appeared to be more radiosensitive than those in the triploid region. Thus, tumors unresponsive to therapy as well as those that recurred after irradiation were mostly triploid. Since most of the tumors had high proliferation rates the proportion of S-phase cells was of only limited value for further classification of the various tumors.In the choice between radical radiotherapy, in which bladder function is maintained, and cystectomy an analysis of tumor ploidy provides at least some basis on which radiation response can be predicted. In the choice of therapy we must also consider the fact that almost 20% of the patients who failed to respond to therapy revealed aneuploidy only in bladder washings after irradiation, indicating existence of concomitant carcinoma in situ.

Tumour‐induced host stromal‐cell transformation: Induction of mouse spindle‐cell fibrosarcoma not mediated by gene transfer

Tumour-induced host-cell transformation has been addressed by examining human tumours in situ and following xenograft to nude mice. We have found evidence for the transformation of host stromal fibroblasts both in vivo and following the introduction of the tumours to in vitro culture. The in vitro culture of one such xenograft--derived from a human prostatic adenocarcinoma--resulted in the outgrowth of a transformed aneuploid mouse cell line. This transformed line was tumourigenic both in BALB/c nu/nu (nude) mice and in heterozygous nu/+mice, with the morphology of a spindle-cell sarcoma. The cell line did not express human isozymes or human histocompatibility antigens, nor were human chromosomes present. Moreover, human DNA sequences were not detected by human Alu repeat sequence element probing in the transformed cell line grown either in vitro or in vivo. The line contained retroviral long terminal repeat sequences but there was no evidence of proviral activation. These findings indicate that tumour cells may cause transformation of neighbouring stromal cells; that this transformation may proceed in the absence of DNA transfer or activation of endogenous proviruses; and that the means of this observed transformation may involve humoral factors elaborated by the tumour cells.

Mitotic rate, DNA distribution, and chromatinin situ sensitivity to heparin in breast cancer

The purpose of this study was to characterize breast carcinomas by cell kinetic parameters. Mitotic rate (MR) and flow cytometrically (FCM) measured cell cycle distribution as well as chromatin testing in situ employing heparin for determination of activated chromatin, provided the following results: MR counted in 73 unselected carcinomas showed an increase up to a tumor size of 4.2 cm (p less than 0.05); beyond this diameter, the MR was found to decrease. In T1-T2 carcinomas, cell cycle stage analysis yielded higher percentages of cells in S and G2M phase for ductal (13% and 12%, N = 22) than for lobular (8% and 7%, N = 8) node-negative carcinomas (p less than 0.002). In ductal carcinomas, lymph node involvement was reflected by higher % G2M values (15%, N = 26) compared with negative cases (12%, N = 22) (p less than 0.05). Ductal node-positive T3-T4 carcinomas (N = 10) revealed a higher % S value (16%) than their T1-T2 counterparts. A correlation between MR and % G2M was established only up to a tumor size of 4.2 cm (r = 0.39, p less than 0.05). A highly sensitive ('H') and a poorly sensitive ('P') subgroup of carcinomas with respect to heparin-induced changes in fluorescence intensity of the G1/0 peak of the DNA aneuploid cell line were identified, as previously shown. These subgroups were here updated with a larger number of carcinomas and were limited to T1-T2 cancers (N = 57). Group 'H' included more younger patients (p less than 0.005), less cases with nodal involvement in ductal carcinomas (p less than 0.05), and lower % G2M values in lobular node-negative cases (p less than 0.05), than group 'P'. DNA diploid cells always existing in DNA aneuploid carcinomas are more sensitive than their aneuploid counterparts (p less than 0.01); however, they strengthen the stratification to 'H' and 'P'. We suggest 'H' carcinomas to be less aggressive than 'P' carcinomas. Small breast carcinomas are recommended to cell kinetic investigations for individualizing adjuvant therapy.

Culture of PNKT-4B cells at invasion permissive and restrictive temperatures. I. Chromosomal analysis

PNKT-4B is an aneuploid cell line derived from a herpesvirus-induced renal adenocarcinoma of Rana pipiens that displays restricted invasion at 21 degrees C or cooler and invasion at 23 degrees C through 28 degrees C. Metaphase chromosomes obtained from subcultures (passages 297; 345-347) grown at 18 degrees C or 28 degrees C were Giemsa stained or N-banded with acidic silver nitrate. Cells grown at 18 degrees C displayed a modal chromosome number of 41, while 28 degrees C cultures displayed a modal number of 40. The distribution of the chromosomes suggests that the two temperatures may be allowing growth of different subclonal populations. N-banding of chromosomes at both temperatures revealed an increase of active nucleolar organizer regions (NORs) over normal frog tissues, 2/2N. Analysis of 200 N-banded spreads from cells grown at each temperature revealed modal numbers of 9 NORs/cell and modal numbers of 6 NOR-containing chromosomes/cell. Nine specific NOR-containing chromosomes were identified and scored. Similar distributions were observed at 18 degrees C and 28 degrees C. The data imply that the modal number of PNKT-4B has shifted since it was first described, 39, and differs at invasion-permissive and -restrictive temperatures. Increased numbers of active NORs and alterations of NOR-containing chromosomes imply an amplification of rDNA over the amount in normal frog.

Prognostic implications of DNA ploidy in squamous cell carcinomas of the tongue assessed by flow cytometry

A total of 47 primary squamous cell carcinomas of the tongue were analysed by DNA flow cytometry. With respect to their clonal DNA content two distinct tumor groups could be distinguished. In 14 cases the tumors (29.8%) were dipoid, whereas in 33 cases (70.2%) additional cell lines characterized by abnormal DNA content could be detected. A significant increase of aneuploid cases with tumor size as well as with decreasing histological differentiation could be detected. Aneuploid cell lines are lacking in T1 as well as in most G1 carcinomas but predominate in T3 and G3. Furthermore, cervical lymph node involvement was recognized in the majority of aneuploid primary carcinomas (81.8%) but was largely lacking in diploid tumors (21.4%). Hence, the presence of aneuploid cell lines is clearly connected with clinical and histopathological parameters, each of which have turned out to worsen the prognosis in tongue carcinomas.