**4.4.4 Unscheduled DNA Synthesis (UDS)**

UDS was detected autoradiographically. 30, 50 or 80 mg/kg in 0.5% DNP was injected subcutaneously into the dorsum of rats [64] and an equivalent amount of saline was injected into rats of the control group. The rats were sacrificed 2 h after injection and small epithelial tissue fragments of the nasal concha, soft palate, esophagus, and forestomach, as well as the basal and lateral side of nasopharynx, were removed immediately. The tissues were processed histologically and autoradiographically. It is easy to identify the nuclei of S phase cells by their extremely heavy labeling. UDS was considered present if there were at least nuclei, each covered by 5-20 silver grains, in a cluster and the number of grains covering the nuclei was no more than that of the background (Fig.4. UDS autoradiograph of nasopharyngeal squamous epithelium**.** DNP was injected subcutaneously into the dorsum

Chemical Carcinogenesis and Nasopharyngeal Carcinoma 75

less differentiated epithelium. In the medial stage, undifferentiated flat cuboid cells located below brain vesicle between the optic cups on both sides of bony plate were designated as primordial respiratory epithelium. The activity of lactic dehydrogenase and its isozymes in nasopharynx during the embryonic and neonatal stages was as follows: In the medial stage, enzymatic activity of the undifferentiated epithelium was clearly higher than that of the late embryonic stage and neonatal rats, but no significant difference was found between the latter two groups. In the nasopharyngeal epithelium of medial, late embryonic, or neonatal stages,

After DNP treatment for 7 months, different kinds of lesions were present in the nasopharyngeal epithelium, such as hyperplasia, dysplasia, carcinoma in situ, and early invasive growth. There were 14 cases of NPC in 24 rats, and some of these had more than one lesion, a total of 22 cancer foci composed of three carcinomas in situ, 14 early infiltrative lesions, and five papillary carcinomas. The histochemical results demonstrated that there was no difference in enzymatic activity of squamous epithelium in normal and experimental groups, while hyperplastic and dysplastic squamous epithelium showed higher enzymatic activity than the normal cells. During these stages, the increased isozymes were mainly of the M-type. In the 22 cancer foci, total activity of lactic dehydrogenase and isozymes (including both M- and H-types) increased to a much higher level than that of the normal tissue, and total activity and isozyme types resembled the undifferentiated cells of the 14- to 17-day-old embryos. At this stage there was also a high level of H-type isozyme activity. Nasopharyngeal epithelium in hyperplastic, dysplastic, or neoplastic stages showed an appreciable increase in the enzymes activity. During the first and second stages, the isozyme was initially mainly M-type, and then H-type isozymes. Since the lactic dehydrogenase in normal nasopharyngeal epithelium was mainly of M-type, the increase in H-type isozyme activity might represent abnormal gene expression during the course of carcinogenesis.

There were two different types of enzymatic change in carcinogenesis course induced by DNP. Lactic dehydrogenase activity increased throughout the stages from hyperplasia and dysplasia up to neoplasia, while acid nonspecific esterase activity significantly increased in the hyperplastic stage, and then decreased or even disappeared in the neoplastic lesions. This suggests that nonspecific esterase is related to the development, diagnosis, and

Esterase activity was histochemically detected in the different sites of nasopharynx, with stronger activity in the base wall than in other sites. The activity was different in various types of nasopharyngeal epithelia; stronger activity was observed in the stratified squamous and transitional epithelium than in the ciliated columnar epithelium, but the intensity was similar in the squamous and transitional epithelium. The deposits of esterase in squamous epithelium were diffusely distributed in the cytoplasm of basal and spinous cells, which were mainly localized at the margin of cilia and cytoplasm of cells. The former were distributed diffusely, while the latter took the shape of dot-like granules. Very weak activity was observed in the goblet cells and the reaction in transitional epithelium was similar to

There were hyperplastic lesions in the squamous epithelium of all animals treated with DNP for 7 months. The enzymatic activity was markedly decreased compared with the surrounding epithelia, squamous epithelium of the control animals, or hyperplasic lesions. No difference was found between dysplasia and neoplasia. In short, enzymatic activity

the isozymes were mainly of M-type.

prognosis of carcinoma.

that in the squamous epithelium.

of rats at 80 mg/kg. The rats were sacrificed 2 h after injection, and the epithelial tissue fragments of nasal concha were immediately removed. The tissues samples were processed histologically and autoradiographically). The results showed that at 4 h after subcutaneous injection of DNP (30 mg/kg), DNA synthesis was inhibited in the squamous epithelia of nasopharynx base and esophagus, both of which are tumor prone [75]. This finding is in agreement with reports of Mirvish [65] et al that some carcinogens inhibited DNA synthesis in their respective target organs. While UDS induction was not detected in the forestomach, there are no references indicating that carcinoma of the stomach was induced by DNP. It was also noted that UDS was autoradiographically present only in the epithelia of organs prone to develop cancer after DNP treatment whereas no fibroblasts in the stroma were positive for UDS. These findings suggest that DNP fails to induce any local and distant sarcoma in the rats, and may be related to selective activation of carcinogens in the target organs epithelia with subsequent DNA damage and repair.

Fig. 4**.** UDS autoradiograph of nasopharyngeal squamous epithelium**.** DNP was injected subcutaneously into the dorsum of rats at 80 mg/kg. The rats were sacrificed 2 h after injection, and the epithelial tissue fragments of nasal concha were immediately removed. The tissues samples were processed histologically and autoradiographically. UDS was considered present when nuclei with 5-20 silver grains UDS positive **(**Original magnification, ×1000**)**

#### **4.4.5 Induction of lactic dehydrogenase, its isozymes, and acid esterase in NPC by DNP**

To investigate enzymatic and isozymatic changes in NPC development, concentrations of urea and pyruvate for the demonstration of H-type and M-type isozymes were determined [76]. The reaction intensity was scored as grade 0-4, depending on the size and color of the formazan granules. The data were statistically analyzed by the ranked data method. There was no significant difference in total activity between squamous and ciliated columnar epithelium, but the reaction in transitional epithelium was more intense. All normal epithelia were mainly of the M-type. In the late embryonic stage, nasopharynx was well developed but covered with

of rats at 80 mg/kg. The rats were sacrificed 2 h after injection, and the epithelial tissue fragments of nasal concha were immediately removed. The tissues samples were processed histologically and autoradiographically). The results showed that at 4 h after subcutaneous injection of DNP (30 mg/kg), DNA synthesis was inhibited in the squamous epithelia of nasopharynx base and esophagus, both of which are tumor prone [75]. This finding is in agreement with reports of Mirvish [65] et al that some carcinogens inhibited DNA synthesis in their respective target organs. While UDS induction was not detected in the forestomach, there are no references indicating that carcinoma of the stomach was induced by DNP. It was also noted that UDS was autoradiographically present only in the epithelia of organs prone to develop cancer after DNP treatment whereas no fibroblasts in the stroma were positive for UDS. These findings suggest that DNP fails to induce any local and distant sarcoma in the rats, and may be related to selective activation of carcinogens in the target

Fig. 4**.** UDS autoradiograph of nasopharyngeal squamous epithelium**.** DNP was injected subcutaneously into the dorsum of rats at 80 mg/kg. The rats were sacrificed 2 h after injection, and the epithelial tissue fragments of nasal concha were immediately removed. The tissues samples were processed histologically and autoradiographically. UDS was

**4.4.5 Induction of lactic dehydrogenase, its isozymes, and acid esterase in NPC by** 

To investigate enzymatic and isozymatic changes in NPC development, concentrations of urea and pyruvate for the demonstration of H-type and M-type isozymes were determined [76]. The reaction intensity was scored as grade 0-4, depending on the size and color of the formazan granules. The data were statistically analyzed by the ranked data method. There was no significant difference in total activity between squamous and ciliated columnar epithelium, but the reaction in transitional epithelium was more intense. All normal epithelia were mainly of the M-type. In the late embryonic stage, nasopharynx was well developed but covered with

considered present when nuclei with 5-20 silver grains UDS positive **(**Original

magnification, ×1000**)**

**DNP** 

organs epithelia with subsequent DNA damage and repair.

less differentiated epithelium. In the medial stage, undifferentiated flat cuboid cells located below brain vesicle between the optic cups on both sides of bony plate were designated as primordial respiratory epithelium. The activity of lactic dehydrogenase and its isozymes in nasopharynx during the embryonic and neonatal stages was as follows: In the medial stage, enzymatic activity of the undifferentiated epithelium was clearly higher than that of the late embryonic stage and neonatal rats, but no significant difference was found between the latter two groups. In the nasopharyngeal epithelium of medial, late embryonic, or neonatal stages, the isozymes were mainly of M-type.

After DNP treatment for 7 months, different kinds of lesions were present in the nasopharyngeal epithelium, such as hyperplasia, dysplasia, carcinoma in situ, and early invasive growth. There were 14 cases of NPC in 24 rats, and some of these had more than one lesion, a total of 22 cancer foci composed of three carcinomas in situ, 14 early infiltrative lesions, and five papillary carcinomas. The histochemical results demonstrated that there was no difference in enzymatic activity of squamous epithelium in normal and experimental groups, while hyperplastic and dysplastic squamous epithelium showed higher enzymatic activity than the normal cells. During these stages, the increased isozymes were mainly of the M-type. In the 22 cancer foci, total activity of lactic dehydrogenase and isozymes (including both M- and H-types) increased to a much higher level than that of the normal tissue, and total activity and isozyme types resembled the undifferentiated cells of the 14- to 17-day-old embryos. At this stage there was also a high level of H-type isozyme activity. Nasopharyngeal epithelium in hyperplastic, dysplastic, or neoplastic stages showed an appreciable increase in the enzymes activity. During the first and second stages, the isozyme was initially mainly M-type, and then H-type isozymes. Since the lactic dehydrogenase in normal nasopharyngeal epithelium was mainly of M-type, the increase in H-type isozyme activity might represent abnormal gene expression during the course of carcinogenesis.

There were two different types of enzymatic change in carcinogenesis course induced by DNP. Lactic dehydrogenase activity increased throughout the stages from hyperplasia and dysplasia up to neoplasia, while acid nonspecific esterase activity significantly increased in the hyperplastic stage, and then decreased or even disappeared in the neoplastic lesions. This suggests that nonspecific esterase is related to the development, diagnosis, and prognosis of carcinoma.

Esterase activity was histochemically detected in the different sites of nasopharynx, with stronger activity in the base wall than in other sites. The activity was different in various types of nasopharyngeal epithelia; stronger activity was observed in the stratified squamous and transitional epithelium than in the ciliated columnar epithelium, but the intensity was similar in the squamous and transitional epithelium. The deposits of esterase in squamous epithelium were diffusely distributed in the cytoplasm of basal and spinous cells, which were mainly localized at the margin of cilia and cytoplasm of cells. The former were distributed diffusely, while the latter took the shape of dot-like granules. Very weak activity was observed in the goblet cells and the reaction in transitional epithelium was similar to that in the squamous epithelium.

There were hyperplastic lesions in the squamous epithelium of all animals treated with DNP for 7 months. The enzymatic activity was markedly decreased compared with the surrounding epithelia, squamous epithelium of the control animals, or hyperplasic lesions. No difference was found between dysplasia and neoplasia. In short, enzymatic activity

Chemical Carcinogenesis and Nasopharyngeal Carcinoma 77

oncogenic ras (a hallmark of DMBA initiation) or TPA exposure induced all CXCR2 ligands. Ras induction of CXCR2 ligands was mediated by autocrine activation of epidermal growth factor receptor and nuclear factor-KB, and potentiated by PKCA. Oncogenic ras also induced CXCR2 ligands in keratinocytes that were genetically ablated for CXCR2. *In vitro*, CXCR2 was found to be essential for CXCR2 ligand-stimulated migration of ras-transformed keratinocytes and for ligand activation of the extracellular signal-regulated kinase (ERK) and Akt pathways. Both cell migration and activation of ERK and Akt were restored by

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been described in a variety of human malignancies and has been suggested to play an important role in carcinogenesis. The epidermis of inducible Stat3-deficient mice treated with 4 hytroxytamoxifen (TM) showed a significant increase in apoptosis induced by DMBA and reduced proliferation following exposure to TPA. In two-stage skin carcinogenesis assays, inducible Stat3-deficient mice treated with TM during the promotion stage showed a significant delay in tumor development and a significantly reduced number of tumors compared with control groups. Inducible Stat3-deficient mice treated with TM before initiation with DMBA also showed a significant delay in tumor development and a

DNP displays some degree of organ specificity for nasopharyngeal epithelium in inducing rat NPC. To clarify the mechanism underlying this DNP organotropic action, a rat NPC model was constructed using DNP, and atypical hyperplasic nasopharyngeal and NPC tissue was obtained from rats at different stages of tumorigenesis. Differential protein expression was screened using proteome analysis and further confirmed by immunoblotting. Expression of heat shock protein 70 (HSP70) and Mucin was increased in the atypical hyperplasia and NPC cells, and we therefore postulated that DNP might upregulate these genes. In further studies to determine whether DNP does regulate HSP70 and Mucin, we treated HENE cells (cultured from biopsies of normal nasopharyngeal tissue) with 2M and 4M DNP and showed that expression of HSP70 and Mucin increased in dose-dependent manner. To confirm the specificity of DNP, we used arsenite as a control because its carcinogenicity has previously been proven [70]. Expression of HSP70 and Mucin was not induced by arsenite. We therefore think that HSP70 and Mucin might be

DNP induced expression of phosphorylated ezrin at threonine 567 (phos-ezrin Thr567) in a dose- and time-dependent manner in 6-10B nasopharyngeal carcinoma cells (Fig.5 Effects of DNP on ezrin phosphorylation at Thr 567**.** 6-10B cells were treated with 2 or 4 M DNP for 24 h (A), and treated with 4M DNP for 12 or 24 h (B), and ezrin and phos-ezrin expression were assayed with immunoblotting). Furthermore, DNP-induced expression of phos-ezrin Thr567 was dependent on increased Rho kinase and PKC activity. The activation of Rho kinase and PKC occurred through binding to Rho kinase pleckstrin-homology (PH) and promotion of PKC translocation to the plasma membrane. Ezrin is associated with induction of filopodia growth in 6-10B cells, and further studies showed that DNP induces filopodia formation in 6-10B NPC cells and also increases invasion and motility of these cells. This indicated that DNP is involved in NPC metastasis, and DNP-mediated NPC metastasis was

significantly reduced number of tumors compared with control groups [69]

**5.3 Biomolecular and signal transduction targeted by DNP** 

specific and important targets of DNP [57].

CXCR2 reconstitution of CXCR2 null keratinocytes [68].

increased in hyperplasia but was decreased in dysplasia and neoplasia, and sometimes even totally disappeared. Similarly, the esterase activity of human NPC was markedly decreased compared with the surrounding epithelia of the cancer foci and the squamous or ciliated columnar epithelia of the nasopharynx in the control cases, and decreased to varying extents in different cancer cells of various cases or in different foci of the same case. The reactivity remained in well-differentiated cancer cells, but entirely disappeared in poorly differentiated cancer cells.
