Perturbation of cell cycle progression in mouse epidermis prior to the regenerative response.

Cell kinetic perturbations that resulted in a wave of increased cell division during the 6-8 hr lag period prior to regenerative DNA replication in mouse epidermis were examined. The epidermis was stimulated to proliferate by adhesive tape stripping, and flow cytometric DNA measurements of isolated epidermal basal cells counting of mitoses, of Colcemid arrested metaphases and of labeled mitoses among basal cells in histologic sections were made. The results showed that mitotic peaks that occur in the prereplicative period subsequent to tape stripping can be explained by a delay in cell progression through the S phase, followed by subsequent release and partial synchrony in further cell cycle progression. Early peaks of mitoses in epidermis stimulated to proliferate should therefore not, without further evidence, be assumed to originate from cells triggered into division from a resting G2 compartment. The results also indicate an initial delayed cell progression through the G2 phase, whereas the mitotic duration seemed to be initially reduced, indicating that the DNA synthesis phase and the G2 phase are parts of the epidermal cell cycle that may be most vulnerable to various types of influences.

Perturbation of cell cycle progression in mouse epidermis prior to the regenerative response.

Cell kinetic perturbations that resulted in a wave of increased cell division during the 6--8 hr lag period prior to regenerative DNA replication in mouse epidermis were examined. The epidermis was stimulated to proliferate by adhesive tape stripping, and flow cytometric DNA measurements of isolated epidermal basal cells, counting of mitoses, of Colcemid arrested metaphases and of labeled mitoses among basal cells in histologic sections were made. The results showed that mitotic peaks that occur in the pre-replicative period subsequent to tape stripping can be explained by a delay in cell progression through the S phase, followed by subsequent release and partial synchrony in further cell cycle progression. Early peaks of mitoses in epidermis stimulated to proliferate should therefore not, without further evidence, be assumed to originate from cells triggered into division from a resting G2 compartment. The results also indicate an initial delayed cell progression through the G2 phase, whereas the mitotic duration seemed to be initially reduced, indicating that the DNA synthesis phase and the G2 phase are parts of the epidermal cell cycle that may be most vulnerable to various types of influences.

Adrenalin has differential effects on epidermal cell cycle progression in mice.

The cell kinetic response after intraperitoneal injection of the 10 micrograms adrenalin was investigated in hairless mouse epidermis. Changes in the proportion of cells in S and G2 phase were studied by means of flow cytometry of isolated basal cells. Changes in the proportion of cells in prophase and metaphase, changes in the mitotic rate (Colcemid method) and in cell cycle progression of 3H-TdR labeled cells were studied in histologic sections. The results showed that adrenalin has a differential effect on cell proliferation in mouse epidermis. The cell progression rate from S phase through G2 phase to metaphase is increased in one cohort of cells shortly after adrenalin injection. Simultaneously another cohort of cells is reversibly delayed or blocked in prophase. In agreement with most previous studies a significantly reduced cell division rate was seen 2-3 hr after adrenalin injection. At this time the proportions of cells in prophase and G2 phase were normalized, whereas a significant increase in the proportion of cells in S phase had cycle progression out of S phase might be responsible for the reduced mitotic rate seen after adrenalin administration.

Evidence of mouse epidermal subpopulations with different cell cycle times.

In order to obtain information on the distribution of total cell cycle times in hairless mouse epidermis, basal cells were isolated and prepared for DNA flow cytometry at intervals after a pulse labeling with 50 microCi of thymidine. The DNA distributions were recorded, and cells were sorted from windows in the S, G2, and G1 phases of the cell cycle, collected on glass slides, and subjected to autoradiography. The proportions of labeled cells were scored in each fraction, and the percentage of labeled mitoses was determined in histologic sections from the same animals. Grain count distributions were recorded at selected time points over labeled cells in sorted fractions and over labeled mitoses. The movement of the labeled S-phase cohort was thus followed through all cell cycle phases. Peaks in labeled cells were observed at about 36 h in S phase, G2 phase, and mitosis, and high levels of labeled G2 cells and mitoses were seen at about 80 h. These results indicate the existence of one rapidly cycling subpopulation of keratinocytes with a cell cycle time slightly less than 30 h, in addition to keratinocytes with considerably longer cell cycle times. The first peak of labeled G2 cells reached only about 30%. This is consistent with earlier findings of about 30% G2 cells with a rapid traverse, and 70% with a considerably delayed traverse through G2 phase. The proportion of labeled G1 cells reached a value corresponding to twice the initial labeling index at 8 h after pulse labeling. This is consistent with previously obtained phase durations, indicating an unperturbed cell cycle traverse of labeled cells from S phase through G2 and mitosis.

Phase I/II study of carboplatin and 5-fluorouracil in patients with advanced head and neck carcinoma.

59 patients with histological verified squamous cell carcinoma of the head and neck, 39 with primary disease and 20 with relapse were given carboplatin and 5-fluorouracil (5-FU) in escalated carboplatin doses. The starting dose with carboplatin was 200 mg/m2 and the dose was escalated to 300 mg/m2, 350 mg/m2, 400 mg/m2 and thereafter by 20 mg/m2 per step. All patients received a dose of 1000 mg/m2 5-FU as a continuous infusion for 5 days. The myelotoxicity was moderate. No patients had grade 4 haemoglobin toxicity, while 7 patients had grade 3 toxicity. 2 patients had grade 4 leucocyte toxicity and 1 patient had grade 3. 4 patients were observed with a grade 4 platelet toxicity. 2 early deaths occurred at a dose level of 420 mg/m2. 18 out of 39 patients in primary treatment responded while 2 out of 20 patients treated for relapse responded. On the basis of the present study the maximum tolerable dose for carboplatin in combination with 5-FU 1000 mg/m2 is between 350 and 400 mg/m2.

Bleomycin and radiation therapy in squamous cell carcinoma of the upper aero-digestive tract: a phase III clinical trial.

A group of 222 consecutive patients admitted with squamous cell carcinoma of the upper aero-digestive tract were studied in a prospectively randomized and stratified clinical trial. One-half of the patients received bleomycin injected intramuscularly 1 hour before the radiation treatment daily for 5 days a week; the other half received radiation therapy without the added chemotherapy. The total dose of radiation in both groups was about the same, and was given with curative intent even to the patients with advanced tumors who constituted the majority in both groups. Interstitial radiation as boost therapy or surgery was added in patients with residual tumor if the lesions were considered operable and the patient's condition would allow surgery. The addition of bleomycin did not increase the combined local and regional tumor control rates nor did it improve the survival, but did significantly increase the morbidity and the complication rate.

DNA flow cytometry in human testicular cancer.

DNA flow cytometry revealed aneuploid tumour stemlines in 19 of 20 primary testicular cancers without significant difference of the ploidy values between seminomas and non-seminomas. In 7 of 8 analyzable histograms the S-phase activity was 22-51%. A metastatic mature teratoma had 6% cells in S-phase. These results support the clinical observation that testicular cancer is usually a rapidly growing human tumour. The high percentage of aneuploidy in testicular cancer may be of clinical value in the diagnosis of this malignancy.

A randomized study evaluating radiotherapy versus chemotherapy in patients with inoperable non-small cell lung cancer.

A combination of cisplatin (70 mg/m2 i.v. day one) and etoposide (100 mg/m2 i.v. day one, 200 mg/m2 orally days 2 and 3) repeated every third week to a maximum of 4 cycles were compared with high voltage radiotherapy, 42 Gy given in 15 fractions over a 3-week period to patients with inoperable non-small cell lung cancer (a shield was used in the posterior field to reduce the total spinal dose less than 40 Gy). One hundred and eighteen patients received radiotherapy; the median survival was 10.6 months compared to 10.5 months for the 116 chemotherapy patients (p = 0.81). The objective response rate (CR + PR) was 42% for the radiotherapy and 21% for the chemotherapy group (p = 0.009). At progression it was optional to cross over to the other treatment modality or to receive phase II chemotherapy. Thirty patients primarily treated with radiotherapy and 54 allocated to chemotherapy received second line antineoplastic treatment.

DNA flow cytometric values in bladder carcinoma biopsies obtained from fresh and paraffin-embedded material.

DNA-histograms were obtained by flow cytometry (FCM) of nuclei prepared from fresh bladder carcinoma biopsies and from 100 micron sections of paraffin-embedded material from the same biopsies. Linear regression analysis of ploidy values from fresh material versus those from paraffin blocks showed excellent correlation (R2 = 0.957). Owing to the high background, the paraffin-embedded material was less suited for analysis of S-phase fraction. It is concluded that DNA FCM of nuclei obtained from paraffin-embedded bladder carcinoma biopsies yields reliable results concerning ploidy, but does not permit evaluation of the S-phase fraction.

Circulating secretory component in breast neoplasms.

The serum concentrations of IgAp and IgMr associated secretory component (SIgA and SIgM) of 98 patients with neoplasms of the breast were measured. Of the 56 patients with carcinomas, 11 had increased concentrations of circulating SIgM, which was almost twice as sensitive as SIgA as a marker for carcinoma. Concentrations of circulating SIgA and SIgM were independent of expression of secretory component, IgA, and carcinoembryonic antigen (CEA); histological tumour grade; and tumour cell DNA ploidy, whereas a weak correlation between SIgA and SIgM and circulating CEA was seen. The three patients who had liver metastases indicated had particularly high concentrations of circulating SIgA and SIgM, whereas no difference was generally seen between patients with malignancy and those with benign tumours.

Establishment of a human meningeal cell line in culture.

A human metastatic meningiosarcoma has been established as a cell line as follows: At autopsy performed 12 hours after death, tumour nodules from the left parietal pleura were obtained under sterile conditions, minced and inoculated into culture vessels. The cells obtained were cloned, and clones with abnormal chromosome stemlines ascertained by G-, C-, and N-banding techniques were then injected subcutaneously into nude mice of the NMRI and BALB/c strains. Tumour nodules from the athymic mice were also minced, established as cell cultures and maintained for further studies. In vitro cytogenetical analysis showed variations in chromosome numbers per cell ranging from 23 to 126; whereas flow cytometric measurements of DNA-specific fluorescence during exponential cell growth in vitro demonstrated the presence of 3 well defined DNA classes around 3C, 6C, and 12C. The xenografts still retained the particular morphological and structural characteristics of the primary tumour, as analyzed by histological sections. The permanent growth integrity of the established meningiosarcoma should render its utilization as a model system for biological studies possible.

Flow cytometry (FCM) of human epidermal cells. A preparation method for epidermal cells and demonstration of circadian variations in the proportion of S-phase cells.

A suction blister technique on the abdominal skin to separate pure human epidermis from its basement membrane is described. After incubation with proteolytic enzymes (trypsin, pepsin) and mechanical shaking, isolated cells and nuclei were stained with ethidium bromide. DNA-specific fluorescence was measured in a flow cytometer using UV excitation light. Cell-cycle phase distributions were obtained from the resulting histograms using a planimetric method. The proportion of cells with an S-phase DNA content showed circadian variations with peak values shortly before noon, and lower values during the rest of the 24 h period.

DNA flow cytometry of cells obtained from old paraffin-embedded specimens. A comparison with results of scanning absorption cytometry (a methodological study).

Suspensions of single cell nuclei were obtained from 31 different samples of 7-9 years old paraffin-embedded bladder cancer biopsies. The DNA content of the ethidium-bromide-stained nuclei was analyzed by flow cytometry (FCM). The tumour stemline ploidy, as determined by FCM, was compared with that obtained by Feulgen scanning absorption cytometry (SACM) in imprints obtained from the same biopsy before it was fixed. Fifteen tumours that were diploid by FCM were also diploid by SACM. All of the 16 tumours with non-diploid DNA-stemlines by FCM were non-diploid by SACM, though minor differences between the ploidy values were occasionally seen when the results of the two methods were compared. The background activity due to cell debris was considerable and resulted in a mean variation coefficient (CV) of 5.1% for human spleen cells fixed and embedded before preparation for FCM in the same way as the tumour samples. In the tumour samples there was a large and unpredictable variation of the ratio between the DNA values of chicken erythrocytes (internal standard) and diploid cells. In some specimens this ratio would have resulted in incorrect evaluation of the FCM DNA histogram. The following method for evaluation of FCM histograms is therefore proposed: Single peak histograms obtained from paraffin embedded tissue should always be interpreted as representative for a diploid cell population. In FCM histograms from paraffin-embedded tissue with more than one peak, the first peak should be considered as representing diploid cells.

DNA flow cytometry in human bladder carcinoma.

The cell kinetic fractions (G0/G1; S; G2 + M) were evaluated by DNA flow cytometry (DNA FCM) in 102 biopsies from bladder carcinoma, previously untreated by cytotoxic therapy, and in 25 biopsies taken at least 3 months after prior treatment (chemotherapy, radiotherapy, surgery). Non-diploid DNA-stemlines were most often found in tumours of a high T category and of a high histopathological grade. Also the number of tumours with a fraction of cells in S-phase above 10% correlated with the clinical stage and histological grade. When the cytotoxic treatment preceded the actual biopsy by 3 months or more the distribution of stemline ploidies in the recurrent or residual tumours were similar to that seen in previously untreated patients. Furthermore, 4 of 5 individual muscle infiltrating bladder tumours treated with surgery, radiotherapy or systemic chemotherapy had the same stemline ploidy before and after treatment. The analysis of ploidy and cell kinetic parameters obtained from DNA FCM offers a possibility to evaluate the prognosis and the therapy effects in human bladder carcinoma.

Internal cysts and fistulae of branchial origin.

Cysts and fistulae related to the second and third pharyngeal pouches are described. These anomalies probably occur more often than presumed. Unilateral recurring infections of the tonsillar region, including parapharyngeal abscess, may be caused by anomalies related to the second pouch. Third pouch anomalies are located in the piriform recess. Cysts may reach a size that causes obstruction to the larynx and hypopharynx, while diverticulae cause few symptoms.

Flow cytofluorometric analysis of a human aneuploid cell line growing in vitro and in intraperitoneal diffusion-chambers.

An aneuploid established cell line originating from human skin (NCTC 3075) was cultivated in vitro culture and in intraperitoneal diffusion chambers in hamsters. In in vitro cell culture a near tetraploid cell line dominated. Shortly after implantation into the diffusion chambers in the peritoneal cavity of hamsters a selective lysis of cells with near tetraploid DNA content occurred, with a relative increase of a diploid subline. After 5 days in the hamster a tetraploid cell line again dominated as in in vitro culture. The use of flow cytofluorometry for ploidy analysis and changes in cell cycle traverse is demonstrated. The possible use of this model system in studies of response to therapy is discussed.

Toxicity, physical function and everyday activity reported by patients with inoperable non-small cell lung cancer in a randomized trial (chemotherapy versus radiotherapy).

In a randomized trial, patients with inoperable non-small cell lung cancer with limited disease were randomly given either radiotherapy (42 Gy) or combination chemotherapy with cisplatin, 70 mg/m2, and etoposide, 100 mg/m2, given every third week with a maximum of 4 cycles. The patients were asked to fill in a questionnaire concerning psychosocial well-being, medical and treatment related symptoms, physical function and everyday activity. Of the chemotherapy patients 61% reported nausea 5 weeks after their last chemotherapy session and 44% had spells of vomiting. Only 14% of the radiotherapy patients had nausea and 5% vomited 14 weeks after start of treatment. Of the radiotherapy patients 64% experienced dysphagia compared to 8% of the chemotherapy patients 6 weeks after the start of treatment.

Epidermal cell kinetics in hairless mice after bleomycin. II. Perturbations after multiple doses.

Bleomycin (BLM) at doses of 0.1 mg/animal was injected into hairless mice every 4 hours for 4 days. Epidermal cell cycle phase distributions were determined by flow cytofluorometric DNA measurements. Labeling indices and mean grain counts were determined by means of autoradiography, and mitotic rates were determined by demecolcine arrest of metaphases. A small, partially reversible accumulation of cells with G2 phase DNA content was observed subsequent to BLM administration. Most of the cells accumulated in G2 phase, however, seemed to stop proliferation and die in this phase. Thus, BLM does not fulfill the criteria for a good synchronizing agent in the hairless mouse epidermis. Flow cytofluorometry alone cannot determine whether piling up of cells in a particular cell cycle phase is true synchrony or an accumulation of lethally damaged cells. Several cell kinetic methods should be used to demonstrate real synchrony.

Epidermla cell kinetics in hairless mice after bleomycin. I. Perturbations after different single doses.

Three different doses of bleomycin (BLM) (2,1, and 0.01 mg/animal) were injected ip into groups of hairless mice. Cell kinetic alterations in the basal layer of the epidermis were studied up to 10 days after BLM administration. The two highest doses of BLM affected the epidermal cell kinetics in a similar way but with different time courses. Only a moderate depression of the labeling index was observed during the first 24 hours. The results indicate that cells affected by BLM in the middle of the G1 phase are arrested in their subsequent S phase, and that thereafter an increase in the rate of DNA synthesis may occur in the arrested cells without increasing the number of cells in S phase. The percentage of cell accumulated in S and G2 phases as determined by flow cytofluorometry never exceeded 130% of control values with any of the doses. Phase durations were determined from percentage of labeled mitoses curves after the highest dose of BLM and were not significantly different from normal values, indicating that the accumulated cels are probably out of cycle and unable to proliferate again. Calculations based on cell counts and mitotic rate showed that the cells had a prolonged lifespan. No block and subsequent release of cells specifically caused by BLM were observed. BLM has a complex effect on the epidermis, and therefore does not seem to be a promising agent for cell synchronization as a basis for combination chemotherapy in vivo.

Survival of large bowel carcinoma patients with different DNA ploidy.

One hundred patients operated for large bowel carcinoma were divided into a distinct aneuploid group of 63, and a near diploid one of 37. Flow cytometry was used for determination of the DNA ploidy pattern. All tumours in the aneuploid group contained one or more aneuploid cell populations. All patients were followed clinically from 3.5 to 7.8 years. The corrected 5 year survival was 64% and 49% for patients with near diploid and aneuploid tumours, respectively (not significant). Significant differences in corrected survival time were not observed for Dukes' stages A, B, and C patients pooled, nor for Dukes' stage D patients. However, for Dukes' stage C patients alone, there was a tendency (P = 0.10) for patients with near diploid tumours to show a better survival. A highly significant predominance of aneuploid tumours was seen in males, in contrast to an equal distribution of aneuploid and near diploid tumours in females. A slight predominance of aneuploid tumours in the left colon and rectum was seen. Both these findings indicate the influence of environmental factors (hormonal, anatomical, phenotypical) on the development of tumours with a particular DNA ploidy pattern.