W A Linden's research while affiliated with University of Hamburg and other places

Ploidy and cell-cycle stage were determined by flow cytometry (FCM) in 46 human renal carcinomas. Cell populations with aneuploid DNA were detected in 46% of these. In the investigated samples, the fraction of cells with abnormal DNA content varied from 8 to 100%. The proliferative activity was generally low as indicated by the small fractions of cells in S and (G2 + M) phases. This was confirmed by the labelling indices on autoradiographic slides. The fraction of cells in phases S and (G2 + M) for tumours that were pre-irradiated with 15 or 25 Gy before nephrectomy was only slightly less than in unirradiated tumours. Comparison of the FCM ploidy with the results of histological grading showed that all cases classified as the most malignant grades IV or IIIB (according to the nuclear and to the combined grading system of Syrjänen and Hjelt (1978) were hyperdiploid. On the other hand, 45% of the hyperdiploid and 89% of the diploid tumours were of the low grades I and II. After a follow-up for 6 months to 2 years, 8/17 patients with hyperdiploid and only 1/14 patients with diploid tumours have died or relapsed with multiple metastases. The results indicate that the aneuploidy of tumours, measured by FCM, might provide useful additional information for prognosis.

Individual properties of gynecologic specimens can produce artifacts in flow cytometric (FMC) measurements, possibly leading to false interpretations. An identification of such artifacts was undertaken by parallel FCM and microscopic investigations. One hundred fifty unselected cervical smears were measured by FCM using the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) for DNA and sulforhodamine (SR 101) for protein. Microscopic specimens were stained by the Papanicolaou technique, and a detailed cytogram was prepared from each smear. FCM discrimination of fluorochrome-stained superficial and intermediate cells was very difficult. On the other hand, a correlation could be established between the fraction of cells from the deeper epithelial layers in the microscopic cytogram and the mean protein content in the FCM histogram. Furthermore, the role of microorganism could be elucidated. Some microorganisms may produce a reduction of the protein content by cytolytic changes. Other microorganisms adhere to the cell surface, resulting in a misleading increase of the DNA fluorescence. Implications for the problem of false alarms are discussed.

Tissues of human renal cell carcinoma and of its lymph-node metastases were transplanted between the 1st and the 4th passage on 80 nu/nu mice. Growth of tumor was observed in all cases. Chromosomal analyses proved the tumor origin of the transplants. The primary tumor showed two histological patterns: a granular solid cell carcinoma and sarcomatoid elements. The lymph nodes showed only sarcomatoid tissue of the primary tumor. Light-and electron-microscopic examinations showed that the transplanted tumor contained only the sarcomatoid tissue of the original tumor. The solid carcinomatous tissue was not found and, therefore, probably not transplanted. Flow cytometric investigations of the primary tumor revealed two cell populations with a DNA content of 6 and 10.8 pg. Both cell colonies were also discovered in the transplanted tumors as well. The transplants of the primary tumors grew significantly slower than those of the lymph-node metastases. After an initial inert phase, the transplanted tumors grew more rapidly in the male than in the female animals. Neither hematogenous nor lymphatic metastases were observed. After local excision, recurrent tumor development was found in 80% of the experimental animals.

Tissues of human renal cell carcinoma and of its lymph-node metastases were transplanted between the 1st and the 4th passage on 80 nu/nu mice. Growth of tumor was observed in all cases. Chromosomal analyses proved the tumor origin of the transplants. The primary tumor showed two histological patterns: a granular solid cell carcinoma and sarcomatoid elements. The lymph nodes showed only sarcomatoid tissue of the primary tumor. Light-and electron-microscopic examinations showed that the transplanted tumor contained only the sarcomatoid tissue of the original tumor. The solid carcinomatous tissue was not found and, therefore, probably not transplanted. Flow cytometric investigations of the primary tumor revealed two cell populations with a DNA content of 6 and 10.8 pg. Both cell colonies were also discovered in the transplanted tumors as well. The transplants of the primary tumors grew significantly slower than those of the lymph-node metastases. After an initial inert phase, the transplanted tumors grew more rapidly in the male than in the female animals. Neither hematogenous nor lymphatic metastases were observed. After local excision, recurrent tumor development was found in 80% of the experimental animals.

In a randomized study of human renal adenocarcinomas the tumours were either preirradiated with 25 Gy of 42 MeV X-rays or removed without pretreatment. Nephrectomy was performed 3 1/2 weeks after the end of irradiation or in the untreated series immediately after diagnosis. The impairment of cell proliferation after irradiation was determined by flow cytometry (FCM) and 3H-autoradiography after in vitro incubation. From FCM DNA distributions the fractions of cells in the phases of cell cycle were computed. The results show good agreement between the S-phase fractions determined by FCM and autoradiography. Preliminary data from 14 patients, 5 preirradiated and 9 unirradiated show that the fraction of cells in S-phase was reduced from 0.06 +/- 0.03 in the unirradiated patients to 0.01 +/- 0.01 in the irradiated ones. 12 of the 14 adenocarcinomas had diploid DNA content (2C) and 2 were hyperdiploid. The results demonstrate that FCM can supplement the techniques available for the assessment of radiation response of human tumours.

One-parameter (nuclear DNA) and two-parameter (nuclear DNA and protein or cellular light scatter) measurements of cervical smears were performed using an ICP 11 and a cytofluorograf 4800 respectively. A total of about 1000 cases was analyzed. For the estimation of nuclear DNA alone two fluorochromes were tested (ethidium bromide (EB) and mithramycin (MMC)) combined with three different methods of cell preparation. For the two-parameter measurements cells were double stained with EB and fluorescein isothiocyanate (FITC). Red fluorescence (EB) versus green fluorescence (FITC) or red fluorescence versus scatter were recorded. A computer analysis of the one-parameter histograms was performed using discriminant analysis and the results were compared with the cytodiagnosis of microscopic specimens stained with the Papanicolaou technique. The error rates of the flow cytometric (FCM) data were as follows: (a) standard EB staining, 11% false negative, 26% false positive, 6% unsatisfactory results; (b) pepsination of vital cells and EB staining, 12% false negative, 14% false positive and 4% unsatisfactory results; (c) MMC staining, 10% false negative, 65% false positive and 5% unsatisfactory results. Our two-parameter measurements prove that, as confirmed by cell sorting, red fluorescence versus scatter allows separation of at least three subpopulations in most analyzed samples: (a) anucleated cells; (b) leukocytes; and (c) intermediate and superficial cells.

Flow cytometry was used for a quantitative study of the accumulation of cells during the radiosensitive G2 phase of the cellular cycle, after exposure to acromycin, bleomycin, cytosine arabinoside, daunomycin, n-hydroxyurea and irradiation with 200 kVp. In cultures of L-cells, the proportion of G2 cells could be increased by the various methods of treatment to 70%, sometimes over 80%. In solid tumors, the largest proportions of G2 can be achieved by local roentgen irradiation, but more than 50% is never obtained. Cytostatic treatment increases the proportion of G2 in various animal tumors by only 2 to 19%. It is concluded that the accumulation of G2 cells that can be achieved in human tumours is not sufficient to affect radiotherapy.

Mouse fibroblasts, subline L-929 F were synchronized by mitotic detachment. The synchronized cell cultures were irradiated with 200 kVp X-rays at different time after mitosis, and age reponse functions and dose effect curves were determined using the colony test. The cell age in the mitotic cycle was obtained from a computer analysis of flow cytometric DNA histograms. Both intrinsic radiosensitivity 1/D 0 and extrapolation numbern were found to vary during the cell cycle. TheD 0 has a maximum value of 176 ± 1 rad in the middle ofG 1 phase and a minimum of 71 ± 1 rad at theS/G 2 transition, while the extrapolation number is rather constant from the beginning ofG 1 phase (1.9 ± 0.1) to the middle ofS phase (2.3 ± 0.1) and reaches a steep maximum of 9.3 ± 1.1 atS/G 2 transition. The values ofn in the various phases of cell cycle are compared with the respective values of the recovery factorγ determined after fractionated irradiation. - Cell survival after a single dose of 616 rad has minima for irradiation atG 1/S transition and in earlyG 2 phase; the survival in earlyG 2 being about 40 times smaller than in earlyG 1 phase. Implications for a cell cycle specific therapy are discussed.

The combined action of Daunomycin and irradiation was investigated using mouse L-929 cells in culture. Survival of cells was measured with the colony assay. Sedimentation in alkaline sucrose gradients was used to study repair of DNA single-strand breaks (SSB) in the presence of various concentrations of Daunomycin. A small increase in radio-sensitivity, as measured by decreasing Do, was obtained for doses of Daunomycin that are considerably toxic to the cells (0.1 microgram/ml). However, the Dq values remained constant even at high concentrations indicating that Daunomycin does not interfere with recovery processes. The rate of rejoining of SSB remained constant up to 1.0 microgram/ml, whereas concentrations of Daunomycin as high as 10 microgram/ml reduced the velocity of repair by a factor of 13. Our data show that concentrations of Daunomycin similar to those required for other DNA-binding drugs are required to inhibit SSB repair. For clinical purposes, no increase in tumour-killing efficiency may be expected from a combined treatment with Daunomycin and radiation.

The proliferation parameters of the Walker carcinoma were estimated from both in vivo and in vitro measurements. tthe transplantable Walker carcinoma 256 was grown in male inbred BD1 rats. During exponential growth, 5--6 days after transplantation, a PLM curve was performed, yielding estimates of TC approximately equal to 18-0 hr, TS approximately equal to 6-4 hr, TG2+M approximately equal to 4-1 hr. With the double labelling technique in vitro under 2-2 atm oxygen we obtained: TC approximately equal to 18-2 hr, TS approximately equal to 8-2 hr, TG2+M approximately equal to 2-0 hr. From pulse cytophotometry DNA content histograms the fractions of cells in the cell cycle phases were calculated using a computer program: fG1 approximately equal to (47-6 +/- 1-1)%, fS approximately equal to (34-1 +/- 1-0)%, fG2+M approximately equal to (18-3 +/- 1-5)%. These fractions remained constant between the fifth and the twelfth day after transplantation. At that time the tumour growth had already slowed down appreciably. The growth fraction determined by repetitive labelling was 0.96 on the fifth and 0-93 on the seventh and eleventh day. The cell loss factor was phi approximately equal to 17% during exponential tumor growth and increased to about 100% between the tenth and twelfth day. The agreement of the cell kinetic data determined by autoradiography from solid tumours in vivo (PLM, continuous labelling) and autoradiography as well as pulse cytophotometry from in vitro experiments (excised material) was satisfactory.