Human Leukocyte Test System Research Articles

Induction of sister chromatid exchanges by cyclophosphamide in human lymphocytes with metabolic activation.

Human leukocytes were treated with CPM for the establishment of the in vitro activating system to detect inactive mutagenic chemicals in the presence of S-9 mix. CPM caused a maximum increase in SCE-frequency after 60min incubation. Further, the frequency of SCEs was markedly increased by additional doses of CPM. It thus appears that chemicals requiring metabolicactivation induce SCEs in the human leukocyte test system.

The human leukocyte test system. VIII. DNA synthesis and mitoses in 3-day cultures stimulated with pokeweed mitogen.

In human leukocyte cultures set up with Ham's F-10 medium and stimulated with pokeweed mitogen (PWM), DNA synthesis and mitotic indices were analyzed by means of (3H)TdR autoradiography and cell counting. The results show a very similar pattern of DNA synthesis and mitotic indices to that found in cultures set up with TC medium 199 and stimulated with phytohemagglutinin (Obe et al., 1975 a).

The human leukocyte test system. X. higher sensitivity to X-irradiation in the G0 stage of the cell cycle of early as compared to late replicating cells.

Leukocyte cultures were set up with X-irradiated whole blood (200 R). Cells starting with their DNA synthesis between 25 and 35 h after cultureinitiation ("early replicating cells") were pulse-labeled with tritiated thymidine ([3H]TdR). Mitoses were collected with colcemid in adjacent intervals from 36 up to 72 h after culture initiation. At fixation times of 50, 56, 62, and 72 h enough mitoses for a determination of the frequencies of chromosomal aberrations (dicentric and ring chromosomes) were found. After that the preparations were processed for autoradiography. All mitoses analyzed for chromosomal aberrations were re-analyzed for labeling, and the frequencies of chromosomal aberrations in labeled (="late replicating cells") mitoses were compared. At all fixation times, higher frequencies of dicentric chromosomes were found in labeled as compared to unlabeled mitoses, indicating a higher sensitivity of early replicating cells to X-irradiation in the G0 stage of the cell cycle.

The human leukocyte test system: IV. The RNA-synthesis pattern in the first G 1-phase of the cell cycle after stimulation with PHA

The human leukocyte test system: IV. The RNA-synthesis pattern in the first G 1-phase of the cell cycle after stimulation with PHA

The human leukocyte test system IV. The RNA-synthesis pattern in the first G1-phase of the cell cycle after stimulation with PHA

The human leukocyte test system IV. The RNA-synthesis pattern in the first G1-phase of the cell cycle after stimulation with PHA

The human leulocyte test system. VII. Further investigations concerning micronucleus-derived premature chromosome condensation.

Premature chromosome condensation (PCC) from X-ray induced micronuclei shows a dose-effect relationship in human leukocytes in vitro. Preparations at different culture times without colcemide treatment reveal complex variations of the frequencies of micronuclei and PCC correlated with the fixation time. The positions of PCC patches in the metaphase plate and the frequencies of different PCC types (S and G2) ar independent on the X-ray dose. The latter indicates that the slowing down of the micronuclei in the cell cycle, which is the reason for the formation of PCC, may be an outcome rather of a regulatory phenomenon than of an unspecific physiological damage of the chromatin included in the micronuclei. This is especially evident from labeling experiments with tritiated thymidine, showing that the extent of asynchrony between main nuclei and micronuclei is independent on the X-ray dose. Labeling experiments with tritiated uridine reveal a X-ray dose dependent suppression of RNA synthesis in cells with main nuclei and micronuclei. THE S-phase nature of "pulverized" PCC patches could be verified by incorporation of tritiated thymidine in aound 50%. Staining of centromeric heterochromatin in micronuclei reveal a frequency of micronuclei with centromeric heterochromatin resembling the frequency of G2-phase PCC found in mitoses.

The human leukocyte test system: III. Premature chromosome condensation from chemically and X-ray induced micronuclei

Premature chromosome condensation (PCC) was induced in human leukocytes in vitro by a bifunctional alkylating agent 2,5-bis-(methoxyethoxy)-3,6-bis-ethylene-imino- p-benzoquinone (A 139), bleomycin and X-rays. Analysis shows that PCC originates from micronuclei that have been slowed down in their cell cycles in respect to the main nuclei. The mitosing chromatin of the main nuclei induced PCC in the associated micronuclei. Morphologically the PCC was mostly of the S-phase type with the chromatin exhibiting a more of less pulverized appearance; in some experiments this was verified by incorporation of [ 3H]thymidine (TdR). In a few cases the PCC looked like G2-phase PCC with ladder-like chromosomes. G1-phase PCC was not found. PCC of the type described in this work may be used as an indicator of induced lagging of chromatin that can be the result of chromosomal aberrations or of non-disjunction.

The human leukocyte test system. VI. The use of sister chromatid exchanges as possible indicators for mutagenic activities.

The trifunctional alkylating chemical mutagen trenimon increases the frequency of sister chromatid exchanges in human leukocyte chromosomes in vitro, as revealed by a BUdR-Giemsa method. Treatment with lead acetate exhibited negative results in this respect. The use of sister chromatid exchanges as possible indicators for mutagenic activities is discussed.

The human leukocyte test system

In human leukocyte cultures st up with TC medium 199,DNA synthesis and mitotic indices were analysed by means of 3H-thymidine autoradiography and cell counting. DNA synthesis starts at around 28 hrs. The frequencies of labelled cells rise slowly and reach a maximum of around 24%. The first mitoses appear at around 38 hrs but up to 49 hrs only very few mitoses can be seen. After that time the mitotic indices rise and reach values of up to 11% cultivation in the presence of BudR for 72 hrs and staining with Hoechst 33258 stain revealed that first, second and third mitoses occur together in the cultures at this time. Irradiation of whole blood and cultivation for 72 hrs leads to mitoses containing dicentric and ring chromosomes with and without fragments, to interphases with micronuclei, to premature chromosome condensations (PCC) and to polyploid mitoses indicating that at this time first and further mitoses are present.

The human leukocyte test system III. Premature chromosome condensation from chemically and X-ray induced micronuclei

The human leukocyte test system III. Premature chromosome condensation from chemically and X-ray induced micronuclei

The human leukocyte test system: II. Different sensitivities of sub-populations to a chemical mutagen

The human leukocyte test system: II. Different sensitivities of sub-populations to a chemical mutagen

The human leukocyte test system II. Different sensitivities of sub-populations to a chemical mutagen

The human leukocyte test system II. Different sensitivities of sub-populations to a chemical mutagen

The human leukocyte test system I. DNA synthesis and mitoses in PHA-stimulated 2-day cultures

The human leukocyte test system I. DNA synthesis and mitoses in PHA-stimulated 2-day cultures

The human leukocyte test system: I. DNA synthesis and mitoses in PHA-stimulated 2-day cultures

The human leukocyte test system: I. DNA synthesis and mitoses in PHA-stimulated 2-day cultures