Beitr. Path. Bd. 158,241-254 (1976)
Original Papers
The Norwegian Radium Hospital, General Department (Chief: H. Hest) and Norsk Hydro's Institute for Cancer Research, Department of Tissue Culture (Chief: R. Oftebro) and Department of Biophysics (Chief: T. Brustad), Montebello, Oslo 3, Norway
"Non-Condensed" and "Condensed" Chromatin in Transitional Cell Carcinoma of the Human Urinary Bladder1) "Unkondensiertes" und "kondensiertes" Chromatin in Dbergangszellkarzinomen der menschlichen Harnblase S. D. FOSSA and With
2
o. KAALHUS
Figures and 1 Table· Received December
12,
1975 . Accepted March 1, 1976
Key words: "Non-condensed" chromatin (Euchromatin) - "Condensed" chromatin (Heterochromatin) - Nuclear size - Ploidy class - CarcinogenesIs
Summary The area and content of "non-condensed" and "condensed" chromatin in smeared Feulgen-stained malignant urothelial cells were determined by means of scanning-cytophotometry. The results were compared with those from similar measurements of benign human transitional epithelial cells. There was no difference between the relative area and content of "non-condensed" and "condensed" chromatin in cancer nuclei and normal urothelial nuclei as far as nuclei of the same size and ploidy class were considered. Within the same ploidy class the relative area and content of "non-condensed" chromatin increased with increasing nuclear size. As increased nuclear size within the same ploidy class is typical for most cancer cells, cancer specimens therefore contained relatively more "non-condensed" chromatin than normal urothelium. Analogously the relative values of "condensed" chromatin decreased in cancer specimens. Only in high-polyploid cancer cells, which occurred more frequentley in undifferentiated tumours, a slight decrease of the relative area and content 1) This study was supported by grants from Grosserer N. A. Stang's Legat til kreftsykdommers bekjempelse, Elisabeth og Knut Knutsen's O.A.5. fond for kreftforskning and Nansenfondet, Oslo. 16 Beitr. Path. Bd. 158
242 .
s. D. Foss~ and o. Kaalhus
of "non-condensed" chromatin was observed as compared with well differentiated diploid tumour cells. It was in polyploid tumours that the absolute area and content of "condensed" chromatin was increased as compared with diploid normal urothelium. This means that the changes in "non-condensed" and "condensed" chromatin were primarily dependent on nuclear size and total chromatin content and were not found to be a characteristic of cancer nuclei as compared with control nuclei of the same size and ploidy. These findings differ from the results of biochemical analyses of heterochromatin both in cells during carcinogenesis and also in cancer cells, but are in agreement with qualitative and quantitative morphological studies of smeared cancer nuclei.
From a morphological point of view Heitz (I929) defined heterochromatin as that chromatin which appears intensely stained in the interphase nucleus. In contrast to these intensely stained or "condensed" chromatin particles the so-called euchromatin is more diffuse and faintly stained ("non-condensed" chromatin). From the biochemical point of view heterochromatin is defined as "repressed chromatin". Frenster et al. (I963) and Schmid (I967) described it as "genetically inactive" and "late replicating" on the basis of cytogenetic studies. It is at the moment impossible to reconcile the results of biochemical analyses of heterochromatin with morphological studies of "condensed" chromatin and, in addition, the role of heterochromatin in carcinogenesis is still uncertain. The aim of this cytophotometric study was to measure "non-condensed" and "condensed" chromatin in nuclei from transitional cell carcinoma of the human urinary bladder and in nuclei from benign human transitional cell epithelium.
Material and Methods Imprint smears were made on two slides from each of 25 biopsy specimens. All biopsies were taken by transurethral resection from patients with histologically proven transitional cell carcinoma of the bladder. The smears were prepared within 3 hours after the biopsy was taken. The wet smears were immediately fixed in a mixture of methanol (85010), formalin (10010) and glacial acetic (5010) (Bohm et a!., 1968a). After hydrolysis (4 n HCl 100 min 28° C) (Bohm et aI., 1968b) the smears were stained according to Feulgen's method. The stain was prepared ad modum Graumann (1953) using Pararosaniline Base No. 7601 (Merck). One hundred nuclei were examined from each biopsy with the SMP 05 (Zeiss, Oberkochen, W.Germany) (objective: Plan 100/1.25 oil; condensor: LD Plan 40/0.6; measuring field diameter: 0.7 f1m; distance between centres of the measuring fields: 0.5 f1m, wavelength: 570 nm). The microscope was connected with a Wang Desk Computer 720 B. The light fields were selected at random by using
Carcinoma of the Human Urinary Bladder . 243 a grid and all intact epithelial cells were measured in every field. This was done to avoid unconscious selection of nuclei with obviously malignant appearance. The nuclear area of a single cell was called a, while the mean nuclear area of a specimen was defined as A. The total extinction of a Feulgen-stained single nucleus (te) is an expression of the total nuclear DNA-content, measured in arbitrary units. The total nuclear area of all measured nuclei (AlOO) was calculated by counting the number of scanning points between the extinction values 0.03 and 1.3 in 100 nuclei per specimen. Analogously the measured total nuclear DNA-content (TE100) - expressed in arbitrary units - was determined by adding up all extinction values of the different scanning points over the nuclear area in 100 cells. The frequency histograms for all scanning points in a specimen regarding the area (A100) and DNA-content (TE100) were drawn for the different extinct,ion classes (0.03-0.09-0.22-0.45-0.75-1.3) (Fig. I). For each biopsy the frequency histogram for the 100 nuclei Feulgen-DNA-values (te) was drawn as described elsewhere (Fossa, 1975) and the modal DNA-value was calculated as a numerical expression of the DNA-stemline. The cut-off line between the "condensed" and "non-condensed" chromatin was chosen at the extinction value 0.22. Using this discriminator value it was found that the nuclear DNA-content of lymphocytes consists of about 60-80% of "condensed" chromatin which agrees well with results of Frenster (1965), who found 70% heterochromatin in lymphocytes by biochemical analysis. The area and the content of "non-condensed" (anon-cond., tenon-cond.) and "condensed" (acond., tecond.) chromatin were determined for each nucleus. In order to describe the relationship (r) between the areas of nuclear "non-condensed" to "condensed" chromatin quantitatively in normal and cancer nuclei, we took rnon-cond. (a) = allOn-colld.la where anon-condo and a are the area of "non-condensed" chromatin and the total area of each nucleus. Analogously, the relationship (s) was calculated for each nucleus concerning the amount of "non-condensed" chromatin of a nucleus: snon-cond. (a) = tenon-cond./te. The corresponding values for "condensed" chromatin were: rcond. (a )
acond. a
= --- =
tecond. Scond. () a = ---= te
rnon-cond. (a) an d
I -
I -
snon-cond. (a)
The average of r (a) (R) over all cells (N), be they diploid (dipl.) or non-diploid (nondip!.) in one specimen was: Rnon·cond.
=
(Ndip!. Rnon-cond. dip!.
""" (~ ndip!. (a) rnon-cond. (a) dip!. area classes
where Ndip!.
=
+ Nnon-dip!..
Rnon-cond.) / (Ndip!. non-dip!.
+ Nnon-dip!.)
+ ~ nnon-dip!, (a) rnon-cond. (a)) / (Ndip!. + area classes
Nnon-dip!.)
non-dip!.
~ndip!. (a) and Nnon-dip!. = ~non-dipL (a) area
area
classes
classes
were the sum over the diploid and non-diploid cells. The values for the average of Snon-cond. (a) over all cells (Snan-cond.) were calculated analogously for each specimen. The values Rand S obtained by averaging over the area distribution of cells in one specimen were corrected (R', S') using a correction procedure described elsewhere
244 . S. D. Foss~ and O . Kaalhus 2
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Original Papers
The Norwegian Radium Hospital, General Department (Chief: H. Hest) and Norsk Hydro's Institute for Cancer Research, Department of Tissue Culture (Chief: R. Oftebro) and Department of Biophysics (Chief: T. Brustad), Montebello, Oslo 3, Norway
"Non-Condensed" and "Condensed" Chromatin in Transitional Cell Carcinoma of the Human Urinary Bladder1) "Unkondensiertes" und "kondensiertes" Chromatin in Dbergangszellkarzinomen der menschlichen Harnblase S. D. FOSSA and With
2
o. KAALHUS
Figures and 1 Table· Received December
12,
1975 . Accepted March 1, 1976
Key words: "Non-condensed" chromatin (Euchromatin) - "Condensed" chromatin (Heterochromatin) - Nuclear size - Ploidy class - CarcinogenesIs
Summary The area and content of "non-condensed" and "condensed" chromatin in smeared Feulgen-stained malignant urothelial cells were determined by means of scanning-cytophotometry. The results were compared with those from similar measurements of benign human transitional epithelial cells. There was no difference between the relative area and content of "non-condensed" and "condensed" chromatin in cancer nuclei and normal urothelial nuclei as far as nuclei of the same size and ploidy class were considered. Within the same ploidy class the relative area and content of "non-condensed" chromatin increased with increasing nuclear size. As increased nuclear size within the same ploidy class is typical for most cancer cells, cancer specimens therefore contained relatively more "non-condensed" chromatin than normal urothelium. Analogously the relative values of "condensed" chromatin decreased in cancer specimens. Only in high-polyploid cancer cells, which occurred more frequentley in undifferentiated tumours, a slight decrease of the relative area and content 1) This study was supported by grants from Grosserer N. A. Stang's Legat til kreftsykdommers bekjempelse, Elisabeth og Knut Knutsen's O.A.5. fond for kreftforskning and Nansenfondet, Oslo. 16 Beitr. Path. Bd. 158
242 .
s. D. Foss~ and o. Kaalhus
of "non-condensed" chromatin was observed as compared with well differentiated diploid tumour cells. It was in polyploid tumours that the absolute area and content of "condensed" chromatin was increased as compared with diploid normal urothelium. This means that the changes in "non-condensed" and "condensed" chromatin were primarily dependent on nuclear size and total chromatin content and were not found to be a characteristic of cancer nuclei as compared with control nuclei of the same size and ploidy. These findings differ from the results of biochemical analyses of heterochromatin both in cells during carcinogenesis and also in cancer cells, but are in agreement with qualitative and quantitative morphological studies of smeared cancer nuclei.
From a morphological point of view Heitz (I929) defined heterochromatin as that chromatin which appears intensely stained in the interphase nucleus. In contrast to these intensely stained or "condensed" chromatin particles the so-called euchromatin is more diffuse and faintly stained ("non-condensed" chromatin). From the biochemical point of view heterochromatin is defined as "repressed chromatin". Frenster et al. (I963) and Schmid (I967) described it as "genetically inactive" and "late replicating" on the basis of cytogenetic studies. It is at the moment impossible to reconcile the results of biochemical analyses of heterochromatin with morphological studies of "condensed" chromatin and, in addition, the role of heterochromatin in carcinogenesis is still uncertain. The aim of this cytophotometric study was to measure "non-condensed" and "condensed" chromatin in nuclei from transitional cell carcinoma of the human urinary bladder and in nuclei from benign human transitional cell epithelium.
Material and Methods Imprint smears were made on two slides from each of 25 biopsy specimens. All biopsies were taken by transurethral resection from patients with histologically proven transitional cell carcinoma of the bladder. The smears were prepared within 3 hours after the biopsy was taken. The wet smears were immediately fixed in a mixture of methanol (85010), formalin (10010) and glacial acetic (5010) (Bohm et a!., 1968a). After hydrolysis (4 n HCl 100 min 28° C) (Bohm et aI., 1968b) the smears were stained according to Feulgen's method. The stain was prepared ad modum Graumann (1953) using Pararosaniline Base No. 7601 (Merck). One hundred nuclei were examined from each biopsy with the SMP 05 (Zeiss, Oberkochen, W.Germany) (objective: Plan 100/1.25 oil; condensor: LD Plan 40/0.6; measuring field diameter: 0.7 f1m; distance between centres of the measuring fields: 0.5 f1m, wavelength: 570 nm). The microscope was connected with a Wang Desk Computer 720 B. The light fields were selected at random by using
Carcinoma of the Human Urinary Bladder . 243 a grid and all intact epithelial cells were measured in every field. This was done to avoid unconscious selection of nuclei with obviously malignant appearance. The nuclear area of a single cell was called a, while the mean nuclear area of a specimen was defined as A. The total extinction of a Feulgen-stained single nucleus (te) is an expression of the total nuclear DNA-content, measured in arbitrary units. The total nuclear area of all measured nuclei (AlOO) was calculated by counting the number of scanning points between the extinction values 0.03 and 1.3 in 100 nuclei per specimen. Analogously the measured total nuclear DNA-content (TE100) - expressed in arbitrary units - was determined by adding up all extinction values of the different scanning points over the nuclear area in 100 cells. The frequency histograms for all scanning points in a specimen regarding the area (A100) and DNA-content (TE100) were drawn for the different extinct,ion classes (0.03-0.09-0.22-0.45-0.75-1.3) (Fig. I). For each biopsy the frequency histogram for the 100 nuclei Feulgen-DNA-values (te) was drawn as described elsewhere (Fossa, 1975) and the modal DNA-value was calculated as a numerical expression of the DNA-stemline. The cut-off line between the "condensed" and "non-condensed" chromatin was chosen at the extinction value 0.22. Using this discriminator value it was found that the nuclear DNA-content of lymphocytes consists of about 60-80% of "condensed" chromatin which agrees well with results of Frenster (1965), who found 70% heterochromatin in lymphocytes by biochemical analysis. The area and the content of "non-condensed" (anon-cond., tenon-cond.) and "condensed" (acond., tecond.) chromatin were determined for each nucleus. In order to describe the relationship (r) between the areas of nuclear "non-condensed" to "condensed" chromatin quantitatively in normal and cancer nuclei, we took rnon-cond. (a) = allOn-colld.la where anon-condo and a are the area of "non-condensed" chromatin and the total area of each nucleus. Analogously, the relationship (s) was calculated for each nucleus concerning the amount of "non-condensed" chromatin of a nucleus: snon-cond. (a) = tenon-cond./te. The corresponding values for "condensed" chromatin were: rcond. (a )
acond. a
= --- =
tecond. Scond. () a = ---= te
rnon-cond. (a) an d
I -
I -
snon-cond. (a)
The average of r (a) (R) over all cells (N), be they diploid (dipl.) or non-diploid (nondip!.) in one specimen was: Rnon·cond.
=
(Ndip!. Rnon-cond. dip!.
""" (~ ndip!. (a) rnon-cond. (a) dip!. area classes
where Ndip!.
=
+ Nnon-dip!..
Rnon-cond.) / (Ndip!. non-dip!.
+ Nnon-dip!.)
+ ~ nnon-dip!, (a) rnon-cond. (a)) / (Ndip!. + area classes
Nnon-dip!.)
non-dip!.
~ndip!. (a) and Nnon-dip!. = ~non-dipL (a) area
area
classes
classes
were the sum over the diploid and non-diploid cells. The values for the average of Snon-cond. (a) over all cells (Snan-cond.) were calculated analogously for each specimen. The values Rand S obtained by averaging over the area distribution of cells in one specimen were corrected (R', S') using a correction procedure described elsewhere
244 . S. D. Foss~ and O . Kaalhus 2
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J
0)
60.
60.
40.
!g 40.
~
u1
t- 20.
20. 0
0
0.03 0D9 0.22 !l45075 13 Ext.class
0.0.3 0D9 D.22 !l45 0.75 1.3 Ex!. cia..
Sptlcimen no. 4
2 60.
60
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