Expression of apoptosis-related proteins and morphological changes in a rat tumor model of human small cell lung cancer prior to and after treatment with radiotherapy, carboplatin, or combined treatment.
OBJECTIVE
In order to understand the apoptosis pathway in tumor cells, differences in cell morphology and expression of apoptosis-related proteins induced by radiation and/or chemotherapy, were investigated in a rat double tumor model comparing cisplatin-sensitive and -resistant human small cell lung cancer tumors.


METHODS
The cisplatin-sensitive human small cell lung cancer cell line (GLC4) and its cisplatin-resistant subline (GLC4-CDDP) were each injected subcutaneously in a different hind limb of the rat. After 15-17 days, radiation (10 Gy), carboplatin (25 mg/kg, intraperitoneal), combined treatment, or no treatment was administered. In combined treatment, carboplatin was administered 24 h before radiation. Tumors were removed and fixed 72 h after carboplatin or 48 h after radiation. Paraffin-embedded slides were stained with hematoxylin/eosin for morphology. Expression of the apoptosis-related proteins p53, p21, Rb, bcl-2, bax, c-myc, Fas and Fas-L was assessed immunohistochemically.


RESULTS
In the GLC4 tumor, carboplatin treatment increased tumor cell size, tumor cell size heterogeneity, and number of apoptotic tumor cells. After radiation and combined treatment, these changes were more pronounced and multinuclear giant cells were observed. In GLC4-CDDP tumors, minimal changes were detected after carboplatin. Following radiation slight increases in tumor cell size, tumor cell size heterogeneity and number of apoptotic tumor cells were observed. No multinuclear giant cells were seen. After combined treatment, GLC4-CDDP showed cellular changes comparable to those in GLC4 cells after radiation or combined treatment, but no giant cells were observed. Untreated, both tumors were equally positive for p53, bax, Fas, Fas-L, c-myc and negative for Rb. Contrary to GLC4, untreated GLC4-CDDP tumors showed no p21 and bcl-2 expression. After combined treatment, an increase in number of bcl-2 positive cells was found in GLC4-CDDP tumors. No p21 was induced in GLC4-CDDP by any treatment modality. In GLC4 tumors, p21 was induced by radiation alone. No further changes in protein expression were induced by any treatment in GLC4 tumors.


CONCLUSIONS
Following therapy, morphological changes in cell size and cell size heterogeneity were more pronounced in GLC4 than in GLC4-CDDP tumors. However, morphological changes in GLC4-CCDP tumors after treatment indicate that carboplatin plus radiotherapy may be effective also in cisplatin resistant tumor cells. An increase in p21 in GLC4 after radiation might facilitate apoptosis. The increase in number of Bcl-2 positive cells after combined treatment and the consistently negative p21 status after any treatment in GLC4-CDDP may protect these tumor cells from apoptosis as a part of their resistance mechanism to cisplatin.
Fokkema
E
E
Department of Pulmonary Diseases, University Hospital Groningen, The Netherlands.
De Vries
E G E
EG
Groen
H J M
HJ
Meijer
C
C
Timens
W
W
D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D016190:Carboplatin / Q000627:therapeutic use*; D018288:Carcinoma, Small Cell / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D003131:Combined Modality Therapy; D004195:Disease Models, Animal; D019008:Drug Resistance, Neoplasm / Q000502:physiology / Q000528:radiation effects; D006801:Humans; D007124:Immunoenzyme Techniques; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D009363:Neoplasm Proteins / Q000378:metabolism*; D009374:Neoplasms, Experimental / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D011878:Radiotherapy; D051381:Rats; D011919:Rats, Inbred Strains; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D016190:Carboplatin