BLEOMYCIN-SENSITIVITY TEST: APPLICATION FOR HUMAN SQUAMOUS CELL CARCINOMA

W. E. G. MOLLER, DR PROF, R. SCHMIDSEDER, DR DR, H. J. ROHDE, DR, R. K. ZAHN, DR PROF, AND H. SCHEUNEMANN, DR DR PROF

Bleomycin (BLM)-inactivating enzyme activity, which is probably a parameter for the efficacy of this antibiotic in cancer therapy, was determined in biopsies from human carcinomas in the head and neck region. Twenty-three cases were studied. It w a s found that highly differentiated as well as moderately differenti- ated squamous cell carcinomas had low extractable activities of this enzyme, comparably to those found in normal skin tissue. Slightly differentiated as well as undifferentiated carcinomas (only one case) had increased enzyme activity. Parallel experiments estimating the total extractable thiol content in the biopsies gave no obvious correlation. The results are discussed in the light of the reported BLM efficacy in the treatment of differently differentiated squamous cell carcinomas.

CUMW 40:2787-2791, 1977.

LEOMYCIN (BLM), DISCOVERED BY UMEZAWA B et al., 'O has antitumor and antibacterial ac- tivities, and its potent effect on human skin can- cer and Hodgkin's disease is well estab- lished.s,7*21 The molecular mode of action of BLM can be determined partially, as the follow- ing actions of BLM are known: splitting off thymine bases from DNA, causing strand-scis- sions in DNA, inhibition of DNA-dependent DNA polymerases, inhibition of DNA synthesis in uitro and in uivo, and influence on chroma- tin. l6*l6 As outlined in detail elsewhere," the ef- fectiveness of BLM is not correlated directly with the amount taken up by the tissue but with the amount of active BLM accumulated in the tissue. The inactivation of BLM is caused by an enzyme, which is present in high activity in liver and in low activity in skin and lung. If these two parameters, a) enrichment of BLM in a special tissue and b) enzymatic inactivation of BLM in the same tissue, are taken into consideration, a prognosis about the cytostatic activity of BLM in a special tumor seems possible. For determi- nation of the BLM-inactivating enzyme activity,

From the lnstitut fiir Physiologische Chemie, Universitst and Zahn-, Mund- und Kieferchirurgie, Mainz, West Ger- many.

Address for reprints: Prof. Dr. W. E. G. Miiller, Institut fiir Physiologische Chemie, Universits't .Johann Joachim Becher Weg 13, 6500 Mainz, West Germany.

The authors gratefully acknowledge loans from the Acad- emy of Science and Letters, Mainz (Germany) and the Landesversicherungsanstalt Rheinland-Pfalz. They thank H. Mack, Illertissen (Germany), for the gift of Bleomycin.

Accepted for publication April 20, 1977.

two methods have been developed; a ) determi- nation of the antibacterial activity using Bacillus subtilis" and b ) determination of the inhibition rate in the in vitro assay using the DNA-depen- dent DNA polymerase. "J' The first method has been successfully used for determinations in nor- mal as well as in animal tumor systems;10*22 the second method has been hitherto applied for normal animal tissue. I' The results obtained by both methods are almost identical.

In the present study the extractable BLM- inactivating enzyme activities in human tumors has been determined to our knowledge, for the first time by use of the DNA polymerase assay. Due to the reported effectiveness of the BLM therapy for patients with squamous cell carci- nomas in the head and neck region,' we chose these tumors for our studies. The levels of en- zyme activity determined were correlated with the different differentiation types of these carci- nomas. In addition total extractable thiol con- tent of the biopsies was estimated. Twenty-three cases were studied.

MATERIALS AND METHODS

Twenty-three patients with squamous cell carcinoma in the head and neck region were classified according to Broders' and TNM classification. '' The distribution of patients ac- cording to disease status and histologic cell type is shown in Table 1. None of these patients had received chemotherapy before or at the time of the study. Ten healthy donors served as con- trols.

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2788 CANCER December 1977 Vol. 40

Biopsies of about 0.8 g were taken, immedi- ately frozen and stored at -15°C before being assayed. One tenth gram of the material was then taken and the DNA content was deter- mined;' usually 0.3 mg DNA was found in this amount of tissue. Subsequently, samples corre- sponding to 2 mg DNA were taken, frozen in liquid nitrogen, and disrupted (for a 1-minute period) in the frozen state to powder in a Micr- odismembrator (B. Braun, Melsungen, Ger- many). The resulting material was quan- titatively suspended in 20 mM Tris-HC1 (pH 7.3) in a ratio 1 : 1 (w/v) and then homogenized in a Dounce homogenizer by five strokes. The sample was centrifuged in an Eppendorf micro- centrifuge (6 minutes; 12,000 g; 2OC). The supernatant was collected and analyzed for BLM-inactivating enzyme activity as well as thiol content.

The determination of the BLM-inactivating enzyme activity was performed by a modified procedure, described by us earlier. 13*'' Ninety microliters of cell extract (test) or of the above mentioned Tris buffer (control) was supple- mented with 10 pl of a BLM solution to reach a final concentration of 11.5 pg BLM/ml. The assays (tests and controls) were incubated for 60 minutes at 37OC. Then the internal DNA in the extracts was inactivated by digestion with micr- ococcal nuclease; the reaction was terminated by EGTA." During this procedure the BLM concentration in the assays decreased to 10 pg/ml. The active BLM concentration in both the tests and the controls was measured by de- termination of the inhibition of DNA synthesis, mediated by the DNA-dependent DNA poly- merase (pol I ; E. cob; isolated according to Jovin et ~ 1 . ~ ) . The test assay was composed as follows: 50 p1 of reaction solution (consisting of: 30 mM Tris-HCI; pH 8.0; 10 mM MgCla; 7.5 mM dithiothreitol; 5 pg bovine serum albumin; 2 jg native herring sperm DNA/ml; 400 pM each of dATP, dCTP and dGTP; 5 pM [Wl-dTTP with a specific radioactivity of 700 cpm/pmol and 14 units/ml of DNA-dependent DNA poly- merase) and 50 pl of test- or control solution (containing 10 pg BLM/ml). Incubation was performed at 37°C for 30 minutes. At 0 and 30 minutes, one sample each of 40 pl was with- drawn from the incubation assays and the acid insoluble radioactivity was determined, a using GF/C filter discs. The dried filter was counted in a dioxane solution." From the 30-minute in- cubation values, the 0-minute values were sub- tracted.

The BLM-inhibitory potency was determined

in a semilogarithmical which was linear under the conditions used for the present study. In the assays containing no cell extracts the potency of BLM to inhibit DNA polymerase reaction is not affected; the potency of the anti- biotic is reduced in the case of an incubation with cell extracts containing the BLM-in- activating enzyme activity. The activity of the BLM-inactivating enzyme is expressed in inhi- bition rate (IR) equals the value of the inhibi- tion in percent. The specific activity is given as IR/2 mg of tissue DNA. The standard deviation of this determination method is low; with four assays carried out in parallel, the standard de- viation after determination of the active BLM concentration does not exceed 4%."

Thiol content was determined by the method of Ellman. '*29 Fifty microliters of the control-(20 mM Tris-HCI; pH 7.3) or the test solution (Tris-HCI extract from biopsies) were added to 850 pl (6 M urea; 20 mM K-phosphate; 50 mM EDTA; pH 7.3) and 100 pl DTNB reagent (20 mM DTNB dissolved in 20 mM K-phosphate buffer; pH 7.3). The mixture was poured into a photometer cell and there developed for 10 mi- nutes at room temperature and the adsorbance at 412 nm was determined. The extinction coef- ficient was taken to be 13,600 M- ' X cm-'. The results are expressed as pmoles/2 mg tissue DNA.

Particulars concerning substrates (prepara- tion, specific activity, etc.) followed those re- ported. "-'' 5',5'-dithiobis (2-nitrobenzoic acid) (DTNB) was obtained from Serva, Heidelberg (Germany).

RESULTS BLM-Inactivating Enzyme Activities

In normal individuals the extractable BLM- inactivating enzyme activity in skin biopsies from head and neck regions amounts to 4.9 IR/2 mg tissue DNA (Fig. 1 ) . Only in two cases of highly differentiated squamous cell carcinomas could the enzyme activity be determined; the specific activity was found to be around normal (5 IR/2 mg tissue DNA) (Fig. 1). The average value of the enzyme activities in moderately dif- ferentiated carcinomas is somewhat higher (8.2 IR/2 mg). In the case of slightly differentiated or undifferentiated (anaplastic) carcinomas the enzyme activity is increased, drastically, 17 or 28 IR/2 mg.

For the above mentioned experiments, only one piece of tumor tissue from each patient was analyzed for BLM-inactivating enzyme activity with one exception. In the case of patient no. 23

No. 6

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BLEOMYCIN-SENSITIVITY TEST Miller et al.

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FIG. 1 . Summary of RLM-inactivating en- zyme activities in squamous cell carcinomas 5 from the head and neck regions at different n histologic cell type. Cell extracts were assayed g g as described in Methods. Each point repre- ~

sents the activity in a biopsy from one individ- .C .I? ual (the numbers correspond to those in Table * I ) . In the riqht column, values for normal skin are qiven. The specific enzyme activity is given

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three skin biopsies from the same lesion were taken and processed. The determinations re- vealed almost identical values; biopsy no. 1 : 29.5 IR/2 mg, no. 2: 32.8 IR/2 mg and no. 3: 31.1 1R/2 mg tissue DNA.

Amount of Total Thiol Content The values for the content of total (protein-

aceous and nonproteinaceous) thiol in normal as well as in tumor biopsies are given in Fig. 2. A considerable deviation of the values is observed; therefore the average values give only a rough basis for the total thiol contents and ape deter- mined for highly, moderately and slightly differ- entiated, and undifferentiated carcinomas and normal skin to be 1.3, 1.8, 3.1, 2.5 and 1.6 pmoles/2 mg tissue DNA, respectively.

DISCUSSION

The results given in this paper clearly show that on the average, the BLM-inactivating en- zyme activity in highly and moderately differen-

FIG. 2. Content of thiol in squamous cell carcinomas at different histologic cell type. Each point represents the concentration in a biopsy from one individual (see Table 1 ) .

high moderote slight no D i f f erentio t ion

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tiated human squamous cell carcinomas of the head and neck region is in the same range as that in normal skin (around 5-8 IR/2 mg tissue DNA). The activity of this enzyme in slightly differentiated and undifferentiated carcinomas (only one case) is considerably higher (higher than 17 IR/2 mg tissue DNA). Comparable studies are available with different normal mice tissues. "*" The following enzyme activities were found: liver 28.4, testis 29.1, spleen 21.4, lung 8.9, brain 2.4 and skin 1.3 IR/2 mg tissue DNA. These data are in obvious agreement with the determination of Umezawa's group" applying a different test method.

Our experiments described here are a first clue that the level of the BLM-inactivating en- zyme activity in the same type of human tumor can vary according to its stage of differentiation. From the experiments with animal tumor model systems, it seems to be proven that the level of the BLM-inactivating enzyme is highly corre- lated with the BLM efficacy in tumor treatment. Therefore, our results concerning the determi-

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2790 CANCER December 1977 Vol. 40

TABLE I . Distribution of Squamous Cell Carcinoma Patients According to Histologic Type and Disease Status (UICC'') ~~~~

Patient No. TNM classification Localization Type of growth Histologic cell type

1 T 3 2 T 3 3 T2 4 T 3 5 T 3 6 T 3 7 T 3 8 T1 9 T2

10 T 3 11 T 3 12 T 3 13 T 3 14 T 3 15 T 3 16 T 3 17 T2 18 T2 19 T 3 20 T 3 21 T2 22 T2 23 T 3

NO N1 N3 N3 N3 N3 NO NO N3 N3 N1 NO N3 NO N3 N3 N3 NO N2 N3 N3 N1 N3

MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO M1 MO MO MO MO MO MO MO

retroauricular upperjaw floor of the mouth lower jaw alveolar lower jaw alveolar lower jaw alveolar upper j aw palate lower jaw alveolar tongue tongue maxillo-palatine lowerjaw alveolar floor of the mouth lowerjaw alveolar lower jaw alveolar tongue tongue lower jaw alveolar upperjaw floor of the mouth tongue floor of the mouth

endophytic endophytic exophytic exophyt ic exophytic endop h y t ic endophytic exophytic endophytic endophytic endophytic exophytic exophytic endophytic endophytic exophytic endophytic exophytic exophytic ex op h y t ic exop hy tic endophytic endophyt ic

moderately differentiated slightly differentiated moderately differentiated slightly differentiated moderately differentiated moderately differentiated moderately differentiated slightly differentiated moderately differentiated moderately differentiated moderately differentiated highly differentiated moderately differentiated moderately differentiated moderately differentiated highly differentiated moderately differentiated moderately differentiated slightly differentiated slightly differentiated moderately differentiated moderately differentiated undifferentiated

nation of the BLM-inactivating enzyme activity in human carcinomas would indicate that highly or moderately differentiated tumors are more sensitive towards BLM than slightly or undiffer- entiated ones. Clinical data confirm this as- sumption; published data from Ichikawa' and Rygard et al." show higher efficacy of BLM in well-differentiated squamous cell carcinomas. In our own experience with 10 out of the 23 patients, listed in Table 1, who were treated with BLM after taking the biopsy, we also found a strong correlation between the level of the BLM-inactivating enzyme activity in the biopsy and the therapeutic result (Schmidseder, to be published). From these data we believe that it is justified to use the level of BLM-inactivating

enzyme activity in a certain tumor as one pa- rameter for the decision for BLM application in human cancer therapy.

Stimulated by the in vitro observation, demon- strating that sulfhydryl compounds stimulate BLM-caused DNA degradation, l7 as well as by the in vivo finding of a correlation between the level of protein-free thiol content of cells and the BLM sensitivity," we determined the total ex- tractable (protein-free and proteinaceous) thiol concentrations in the different carcinoma biop- sies. The results revealed no striking correlation. I t is the task for the future to check whether probably only the protein-free thiol fraction is one further reliable parameter for a pre- therapeutic sensitivity test in BLM therapy.

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