Impaired cell motility in chronic myeloid leukemic granulocytes related to altered cytoskeletal pattern.

The bactericidal activity of polymorphonuclear leucocyte (PMNL) against infection stimulates cytoskeletal changes accompanied with alteration in adhesion and locomotion. Microfilaments, the motile apparatus is known to regulate these changes by polymerization of monomeric G-actin to fibrous F-actin. PMNL from chronic myeloid leukemia (CML) patients have been reported to be defective in locomotion in response to synthetic peptide, n-formyl-methionyl-leucyl-phenylalanine (fMLP) but the mechanism leading to defective locomotion and their spatial reorganization remains unclear. Therefore, in order to study the cause of defective motility of PMNL from CML patients the spatial distribution and reorganization of microfilaments and microtubules in response to fMLP have been examined by transmission electron (TEM) and scanning electron microscopy (SEM). Under SEM, the PMNL-CML surface appeared smoother with reduced ruffling resulting in rounding off cells with lesser polarized morphology. Unstimulated PMNL from normal as well as CML subjects showed shorter and fewer microtubules and evenly distributed microfilaments as compared to fMLP stimulated PMNL. It is proposed that the cause of defective locomotion was due to reduced surface activity as a consequence of altered cytoskeletal configuration. This phenomenon seems to be related to impaired functional appendages and as a whole led to the defective cell motility and hence reduced chemotaxis in PMNL from CML patients.

Establishment and characterization of normal and initiated hamster tracheal epithelial cell system.

The development and characterization of normal hamster tracheal epithelial (HTE) cell system and its initiated subline is described in the present study. Normal HTE cells grew in a monolayer, had a stable diploid karyotype, were anchorage dependent and non-tumorigenic. The presence of desmosomal attachments and keratin filaments confirmed the epithelial nature of these cells. An initiated subline DTC8 was isolated after treatment of HTE cells with a suboptimal dose of 9,10-dimethyl-1,2-benz[a]anthracene (DMBA). These DTC8 cells grew in a monolayer, had a higher growth rate and saturation density, were weakly anchorage independent and non-tumorigenic. Treatment of DTC8 cells with 100 ng 12-O-tetradecanoyl phorbol-13-acetate (TPA), resulted in transformation of these cells which then showed anchorage independent growth on semisolid agar and formed tumours in 85% animals. As DTC8 cells showed heterogeneity in chromosome number, they were further cloned by the limiting dilution method using gamma-irradiated hamster embryonic fibroblasts as a feeder layer. The clone H7I, isolated among these clones had all the properties of initiated cells.

Chronic myeloid leukemia granulocytes have lower amounts of cytoplasmic actin.

Chronic myeloid leukemia (CML) granulocytes had earlier been shown to be defective in microfilament mediated phenomena such as chemotaxis and fluid phase pinocytosis when compared to normal granulocytes. The present studies were carried out to determine whether a quantitative alteration in actin or a change in isoform status could be responsible for some of the defects. Relative proportions of beta and gamma isoforms were found to be unaltered between normal and CML granulocytes. The amount of actin was significantly lower in CML cells. When the actin was expressed as percent of total protein in the granulocytes, it was found to be significantly lower in CML cells. The lower amount of actin may be responsible for some of the defects seen in CML cells.

Fluid pinocytosis and esterase-oxidase in chronic myeloid leukemic granulocytes are differentially stimulated by chemotactic peptide.

Binding of a chemotactic peptide, n-formyl-methionyl-leucyl-phenylalanine (FMLP) to polymorphonuclear leukocytes (PMNL) leads to a series of biochemical and functional events. We have studied stimulation of fluid phase pinocytosis (FP), esterase and oxidase by FMLP in CML PMNL, by flow cytometry. Stimulation of FP in CML PMNL was lower than that in normal PMNL but, stimulation of esterase and oxidase was comparable to that in normal PMNL. Thus, early response to FMLP which is dependent on the integrity of actin network seems to be defective in CML PMNL.

PMN cells from chronic myeloid leukemia (CML) patients show defective chemotaxis in remission.

The chemotactic index (C.I.) of granulocytes from chronic myeloid leukemia (CML) patients at diagnosis and in subsequent remission was measured using different concentrations of the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), by time lapse cinematography and compared with that of normal granulocytes. The C.I. of CML polymorphonuclear leucocytes (PMNL) at diagnosis and in remission was significantly lower than the C.I. of PMNL from normal subjects (p less than 0.001 and 0.05 greater than p greater than 0.02, respectively). PMNL from CML patients at diagnosis showed increased speed after stimulation with FMLP. In most of the CML patients, the highest values of C.I., speed and the number of motile cells were obtained at FMLP concentrations of 10-100-fold higher than those required for normal PMNL. These results suggest an alteration in the interaction between FMLP and its receptors and that events occurring after FMLP binding are also altered. It was earlier shown that PMNL from CML patients in the active stages of the disease show defective chemotaxis. Present studies show persistence of such defective cells in the peripheral blood of CML patients in remission. These results also suggest that defects in PMNL from CML patients in remission. These results also suggest that defects in PMNL from CML patients may be constitutional.

Flow cytometric studies on actin polymerization in PMN cells from chronic myeloid leukemia (CML) patients.

Studies in our laboratory have shown that polymorphonuclear leucocytes (PMNL) from chronic myeloid leukemia (CML) patients are defective in chemotaxis towards a synthetic peptide, n-formyl-methionyl-leucyl-phenylalanine (FMLP), during the active phases of the disease and in remission. Actin plays a major role in cellular movements and binding of chemo-attractant to cells induces polymerization of G-actin to F-actin. We have, therefore, compared polymerization of actin in FMLP stimulated PMNL from CML patients with those from normal subjects by fluorescence microscopy and flow cytometry, using F-actin specific probe, NBD-phallacidin. Our results show that binding of FMLP to normal PMNL induces rapid conversion of G-actin to F-actin followed by depolymerization to some extent. In CML PMNL, such a biphasic response is not seen. Conversion of G-actin to F-actin is slower and F-actin content is significantly lower than that in normal PMNL. Moreover, organization of F-actin is different in CML PMNL as compared to that in normal PMNL.

Defective chemotaxis and adherence in granulocytes from chronic myeloid leukemia (CML) patients.

Adherence and motility of granulocytes from chronic myeloid leukemia (CML) patients was compared with that of granulocytes from normal subjects. The percentage of non-adherent granulocytes was significantly higher in untreated CML patients and patients in relapse and acute blastic crisis (ABC) (P less than 0.01). Chemotactic Index (C.I.) of granulocytes moving in a gradient of a synthetic chemoattractant F-Met-Leu-Phe was measured by time-lapse cinématography. While 84.5 +/- 3.53% of normal granulocytes were motile, only 30.3 +/- 14.7% granulocytes from untreated patients, 33.8 +/- 21.3% granulocytes from relapse patients and 36 +/- 9.9% granulocytes from ABC patients were found to be motile. The C.I. of motile granulocytes from CML patients was significantly lower in untreated patients (P less than 0.05), in patients in relapse (P less than 0.01) and in patients in ABC (P less than 0.05), as compared to that of normal granulocytes. Visualization of cytoplasmic actin by indirect immunofluorescence, revealed the presence of actin in granulocytes from patients in all stages of the disease. Thus, granulocytes from CML patients were defective in directional locomotion. Organized actin filaments were found in the small percentage of motile cells still found in CML patients.

Karyotypic findings as an independent prognostic marker in chronic myeloid leukaemia blast crisis.

Fifty-three patients with Ph positive chronic myeloid leukaemia in blastic phase were studied. Additional abnormalities were found in 29 (55%) patients and were more common in myeloid (64%) than lymphoid (45%) blast crisis. The most frequent were +Ph (32%), +8 (28%), +19 (19%), +20 (9%) and +21 (9%). i(17q) (9%) was associated with thrombocytopenia (5/5) and basophilia (2/5). The incidence of additional abnormalities was higher in patients treated with busulphan (70%) than hydroxyurea (44%). No significant differences were noted in the mean values of the clinical and haematological findings recorded at blast crisis between patients with only Ph positive (PP) cells and those with additional abnormalities (AP + AA). Univariate analysis identified karyotypic findings as an independent prognostic marker indicating its significance in assessing the response to therapy and survival after the onset of transformation.

Establishment and characterization of four new squamous cell carcinoma cell lines derived from oral tumors.

Four cell lines were established from squamous cell carcinomas (SCC) of the oral cavity. Cell lines AW 13516 and AW 8507 were derived from poorly differentiated SCC and epidermoid carcinoma of the tongue respectively. Cell line AW 10498 was derived from moderately differentiated SCC of the lower alveolus, and AW 9803 grew from a well-differentiated SCC of a retromolar trigone. The cultures showed typical epithelial cell morphology, numerous mitotic figures, occasional multinucleated giant cells, individual cell diskeratosis and nuclear and nucleolar abnormalities. The cell lines AW 13516 and AW 8507 were fast growers with a doubling time of 35.5 h and 31.9 h, respectively, which was independent of the initial seeding density. Cell lines AW 10498 (doubling time 52.2 h) and AW 9803 (doubling time 66 h) showed slower growth and had shorter doubling times at higher seeding densities. The presence of cytokeratins was detected in all the four cell lines by using polyclonal antikeratin antisera in indirect immunofluorescence and in Western blotting. None of the cell lines expressed major histocompatibility complex (MHC) class II antigens. MHC class I antigens were expressed by three cell lines but not by AW 9803. Flow cytometric analysis of DNA content and chromosomal studies suggested the presence of polyploidy and aneuploidy in all the four cell lines. Ultrastructural studies revealed typical epithelial cell features, such as the presence of desmosomes, tonofilaments and keratin bundles.

Studies on sister chromatid exchanges in patients with Hodgkin's disease.

Sister chromatid exchange frequencies were analyzed in bone marrow cells and in stimulated peripheral blood lymphocytes from 20 patients with Hodgkin's disease. Sister chromatid exchange studies were also carried out in the bone marrow of eight and peripheral blood of 11 normal subjects separately. Among the patients, ten were newly diagnosed and ten patients were survivors who had received therapy 30-64 months prior to studies. The mean sister chromatid exchange frequencies in bone marrow and peripheral blood of untreated patients were 3.93 +/- 0.7/cell and 4.09 +/- 0.91/cell, respectively, which were not significantly different from those of normal subjects (3.22 +/- 0.7/cell and 5.16 +/- 1.3/cell in bone marrow and peripheral blood, respectively). The mean sister chromatid exchange frequencies in bone marrow and peripheral blood in five of the untreated patients 3-4 months after initial therapy and in a treated (30-64 months after therapy) group were close to the sister chromatid exchange values of the untreated group. Analysis of the distribution of exchanges per chromosome or chromosome groups showed a nonrandom distribution of exchanges in chromosomes #1, #2, and #3, and in E-, F-, and G-group chromosomes in the normal controls and in Hodgkin's disease patients.

Cytogenetic features of erythroleukemia (EL). A study of 11 cases.

Cytogenetic studies were carried on 11 patients with erythroleukemia (EL). Most of these patients showed major chromosomal abnormalities (MAKA), karyotypic instability, and complex chromosomal rearrangements. On the basis of the cytogenetic criteria, 10 patients could be distinguished into erythroid (9 cases) and myeloid types of EL (1 case). The patients did not show any consistent chromosomal abnormality. However, abnormalities of chromosomes 1, 3, 7, 8, 16, and 17 were seen in more than one patient. In patients with the erythroid type of EL, besides the MAKA pattern, three patients showed increased frequency of hyperdiploid polyploid cells ranging from triploidy to tetraploidy.

Cytokeratin expression in precancerous lesions of the human oral cavity.

Cytokeratin (CK) expression was studied in buccal mucosa (BM) from 20 leucoplakia and 7 submucous fibrosis patients using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and two-dimensional gel electrophoresis with iso-electric focussing (IEF) as the first dimension. Normal BM expresses CK 4, 5, 13, 14 and perhaps 19. Of 20 leucoplakia samples analysed, CK 5 was not detected in 17 samples, while CK 14 was not found in 13 samples. CK 1 and CK 8 were aberrantly expressed in six and seven samples, respectively. CK expression in contralaterally collected uninvolved tissues from 3 patients showed a normal pattern in two samples. Non-expression of CK 5 was observed in five of seven submucous fibrosis samples, while CK 14 was not detected in only two samples. CK 8 was aberrantly expressed in three samples. All the leucoplakia patients were chronic tobacco chewers. Thus, non-expression of CK 5 may be an early event occurring in tobacco-associated pathological changes in the BM.

Terminal differentiation of human leukemic blasts in response to 12-0-tetradecanoyl-13-phorbol acetate (TPA).

Phorbol ester, 12-0-Tetradecanoyl-13-Phorbol-Acetate (TPA), induces a terminal macrophage-like differentiation of cells from human acute myelogenous leukemia cell lines. We report here that blastoid cells obtained from acute nonlymphocytic leukemia (M1-M2) undergo differentiation-related changes characteristic of macrophage lineage after exposure to TPA. Blast cells from a patient with ANLL-M1-M2 underwent morphological, functional and histochemical changes after treatment with 1 x 10(-7) and 1 x 10(-8) M TPA. The changes included adhesion to the plastic substrate, 2-4 fold increase in the number of NBT positive cells and an increase in the number of alpha-naphthyl-acetate esterase (alpha-NAE) positive cells. These differentiation changes after treatment with TPA were followed by decrease in proliferative index and G1 cells containing high RNA as estimated by flow cytometry. Of the thirteen cases of undifferentiated or unclassified leukemias studied, two failed to respond to TPA. These data suggest that leukemic blasts retain their ability to express a variety of differentiated functions on induction by TPA. Our data gives evidence suggesting that the "switch" into the differentiation pathways occurred after inhibition of proliferation and reduction in the percentage of G1 high RNA containing cells.

Effects of an aqueous extract of processed bidi tobacco on the growth of hamster tracheal epithelial cells.

Inhalation of tobacco dust is responsible for elevated genotoxicity and pulmonary ailments in workers engaged in processing tobacco for the manufacture of bidis, the Indian version of cigarettes. Tracheal tissue being the major site of interaction with tobacco dust, the effects of different concentrations of an aqueous extract of bidi tobacco (ATE) on the growth of a hamster tracheal epithelial cell line (HTE) were investigated. Colony forming efficiency assay revealed that ATE was cytotoxic only at the highest concentration of 5.0 mg/ml. In cultures treated with 1.25 mg/ml ATE, the cell doubling time and growth rate were similar to that of the controls, while a significant increase in cell doubling time (29.4+/-0.3 h vs 14.0+/-3.75 h, P<0.001) was observed at 2.5 mg/ml ATE concentration. Exposure of HTE cells to the non-toxic ATE concentration of 2.5 mg/ml was found to stimulate ornithine decarboxylase (ODC) activity, incorporation of [3H] methyl thymidine into DNA and increase in the S phase fraction was seen by flow cytometry. However, a 56% reduction in the growth rate of cultures treated with 2.5 mg/ml ATE was related to the prolongation of the traverse of cells through S phase. ATE-induced growth suppression was reversed when cultures were grown in ATE-free medium or upon repeated exposure to ATE. The findings suggest that increased tracheal cell proliferation induced by chronic inhalation of tobacco dust may contribute to the development of pulmonary disorders and possibly neoplasia in exposed individuals.

Cytokeratin expression in human fetal tongue and buccal mucosa.

Expression of cytokeratins (CK), a subset of intermediate filament (IF) proteins in epithelia, is developmentally regulated. CK expression may also change after malignant transformation. Our earlier studies on CK expression in human oral tumours and pre-cancerous lesions have shown specific changes in CK expression. We analysed CK expression in human tongue and buccal mucosa (BM) in fetuses in the embryonic age group of 16 to 27 weeks using biochemical and immunohistochemical techniques to find out whether there is any similarity in CK expression in human oral squamous cell carcinomas (SCC) and fetal oral tissues. CK 1, 8 and 18 were detected in a majority of samples using both techniques. Our earlier studies had shown aberrant expression of CK 1 and 18 in many of the oral SCC and leukoplakias. Studies by immunohistochemistry showed that these different CK antigens were expressed in different cell layers. CK 1(2) were present in the stratified epithelial layers whereas CK 8 and 18 were restricted to glandular epithelium. Till 27 weeks of gestation, both tongue and BM expressed CK 1, 8 and 18 along with CK 6 and 16. Thus, fetal tissues showed some similarities in CK pattern with their respective SCC.

Cytogenetic studies in Hodgkin's disease.

Chromosome studies were carried out by G-banding technique on the bone marrow cells of 24 newly diagnosed, untreated Hodgkin's disease patients and 25 treated patients. Seven of these treated patients had also been studied at diagnosis. In the untreated group of patients, cytogenetic studies were carried out on stimulated peripheral blood lymphocytes in 11 patients and on skin fibroblasts in five. Of the 24 untreated patients, 14 showed normal diploid pattern, while 10 were seen with 8-30 per cent chromosomally aberrant cells in the bone marrow. The frequent anomalies were trisomy C/8 and trisomy 22 seen in 5 and 4 patients respectively. The cytogenetic picture of peripheral blood lymphocytes revealed normal diploid pattern in 7 patients; while 4 other patients showed abnormal clones with trisomy 21. The cultured skin fibroblasts represented normal diploid karyotypes. An altered karyotypic pattern was seen in the bone marrow of treated patients. In patients with abnormal karyotypes, the common anomalies were monosomy C, monosomy D/15 and trisomy 21. In patients which showed no involvement of the bone marrow by haematological parameters, chromosomally abnormal karyotypes were seen in the marrow. Thus, marrow involvement can be detected earlier cytogenetically.

DNA analysis of breast cancer by flow cytometry & correlation with other prognostic parameters.

Flow cytometric estimation of DNA content (ploidy and S-phase fraction--SpF) was done on breast cancer tissues from 171 patients. Twenty eight per cent of the tumours were diploid and 72 per cent were aneuploid. SpF was measurable in 82 DNA histograms; of these 22.4 per cent had SpF less than 10 per cent, 34.1 per cent had SpF between 10-20 and 43.5 per cent had SpF greater than 20 per cent. The mean SpF of the measurable histograms was 19.01 per cent with a range 1.78 to 45.19 per cent. A significant correlation between DNA ploidy and SpF was observed (P less than 0.01). Eighty nine per cent of diploid tumours had SpF less than 10 per cent and 73 per cent of aneuploid tumours had SpF greater than 20 per cent. A significant correlation was also found between ploidy and SpF and oestrogen receptor (ER) status of the tumours (P less than 0.05) and between SpF and progesterone receptor (PgR) status of the tumours (P less than 0.05), but not between ploidy and PgR status of the tumours. A significant direct correlation was observed between SpF and tumour grade (P less than 0.05), but not between ploidy and tumour grade. No correlation was observed between DNA ploidy and SpF and tumour type, tumour size, axillary lymph node involvement, age and menopausal status of the patients. Although the incidence of breast cancer is one-third of that reported in the Western countries, there is apparently no biological difference between the various parameters studied.

Studies on granulocyte functions in patients with chronic myeloid leukemia.

Granulocyte functions, viz. endocytosis, NADPH oxidase activity and iodination by leukocytes, were studied in granulocytes isolated from 17 chronic myeloid leukemia (CML) patients at initial diagnosis (stage I), from 10 patients in relapse (stage II), and 10 patients in acute blastic crisis (stage III). The mean phagocytic index of granulocytes from CML patients was similar to the normal value. NADPH activity decreased as the disease progressed. Thus, the amount of formazan produced was lower in granulocytes from patients in stage II (P less than 0.05) and stage III (P less than 0.01) than that produced by normal granulocytes. H2O2-Myeloperoxidase-dependent iodination was found to be significantly reduced in granulocytes from all stages of the disease compared to that of normal, stage I (P less than 0.01), stage II (P less than 0.05) and stage III (P less than 0.01). It thus seems that granulocyte function becomes less efficient as the disease progresses towards acute blastic crisis. Immature cells from the same patients carried out these functions at a more reduced level than did their mature counterparts.

Multiparametric evaluation of retinoic acid-induced terminal differentiation of blastoid cells from acute non-lymphocytic leukemia patients in vitro.

Recent findings that retinoic acid (RA) induces terminal granulocytic differentiation of the human promyelocytic leukemia cell line HL-60 in vitro and blast cell maturation in patients suffering from acute non-lymphocytic leukemia (ANLL) prompted an investigation on the ability of this agent to induce terminal maturation in blast cells from ANLL patients in vitro. We tested the ability of RA at 3 x 10(-6) M, 3 x 10(-7) M and 3 x 10(8-) M concentrations to induce differentiation in blastoid cells from 16 patients with ANLL using cytochemical and cytologic parameters, in addition to cytofluorometric methods. Leukemic cells in primary culture from all the patients underwent cytochemical and biochemical changes after treatment with RA. However, the extent of differentiation-positive cell clones (D+ clones) varied from patient to patient. Morphologic maturation was observed in a significant number of bone marrow samples. Leukocyte alkaline phosphatase and NBT reduction ability of cells, which are biochemical markers of granulocytic differentiation, were also significantly increased with a simultaneous decrease in DNA and RNA synthesis (which was estimated using a Phywe ICP-11 impulse flow cytometer).