Human Oral Tumor Research Articles

Differentiation and cathepsin D expression in human oral tumors.

This study aimed to ascertain whether cathepsin D expression could be related to the stage of differentiation of oral tumors. Human oral biopsies of 10 squamous cell carcinomas and of the corresponding perilesional normal tissues were used. The tumors had all been clinically graded as advanced stage but nonmetastatic; five were classified histopathologically as poorly differentiated. The gene expression of cathepsin D and keratin K13 in the biopsies was measured by reverse transcription polymerase chain reaction. Ratios of tumor-to-control readings helped compensate for sample variability. Keratin K13, as a suprabasal cell marker, tended to confirm the histological grading of the tumors (but was not otherwise useful in distinguishing tumors from normal tissue). Substantial overexpression of cathepsin D was found in the poorly differentiated tumors. Cathepsin D overexpression is considered a prognostic indicator of metastasis. In this sample, it was also associated with dedifferentiation. Cathepsin D might serve as a valuable gauge in clinical exploration of the connection between dedifferentiation and metastasis.

Cloning, Mapping, Expression, Function, and Mutation Analyses of the Human Ortholog of the Hamster Putative Tumor Suppressor Gene doc-1

doc-1 is a putative tumor suppressor gene isolated and identified from the hamster oral cancer model. Here, we report the molecular cloning and the functional characterization of the human ortholog of the hamster doc-1 gene. Human doc-1 cDNA is 1.6 kilobase pairs in length and encodes for a 115-amino acid polypeptide (12.4 kDa, pI 9. 53). Sequence analysis showed 98% identity between human and hamster doc-1 protein sequences. DOC-1 is expressed in all normal human tissues examined. In oral keratinocytes, expression of DOC-1 is restricted to normal oral keratinocytes. By immunostaining of normal human mucosa, DOC-1 is detected in both the cytoplasm and nuclei of basal oral keratinocytes; while in suprabasilar cells, it is primarily found in the nuclei. Human oral cancers in vivo did not exhibit immunostaining for DOC-1. Like murine DOC-1, human DOC-1 associates with DNA polymerase alpha/primase and mediates the phosphorylation of the large p180 catalytic subunit, suggesting it may be a potential regulator of DNA replication in the S phase of the cell cycle. Using a human doc-1 cosmid as a probe, human doc-1 is mapped to chromosome 12q24. We identified four exons in the entire human doc-1 gene and determined the intron-exon boundaries. By polymerase chain reaction and direct sequencing, we examined premalignant oral lesion and oral cancer cell lines and found no intragenic mutations.

Transfection and expression of MnSOD cDNA decreases tumor malignancy of human oral squamous carcinoma SCC-25 cells.

Overexpression of human manganese-containing superoxide dismutase (MnSOD) activity has been demonstrated to suppress malignancy in human melanoma and breast carcinoma cells in vitro and in vivo. To study its effects on human oral squamous carcinoma cells, stable transfection and expression of MnSOD in SCC-25 cells have been conducted. The MnSOD-overexpressing cell clones were shown to have approximately two- to five-fold increased MnSOD activity compared to the wild-type parental- or vector control-transfected cell clones, respectively. Plating efficiency with different concentrations of serum was decreased in the high MnSOD activity cell clones. Soft agar assays demonstrated that the clonogenic fractions of high-expressing MnSOD clones were dramatically reduced. When inoculated in nude mice, tumor growth was markedly inhibited in MnSOD overexpressing cell clones compared with the wild-type or vector control transfected cell lines. Thus, gene therapy of human oral cancer by increasing the expression of MnSOD activity in target cells might be used to prevent or reduce human oral tumor malignancy.

Expression of type IV collagen-degrading metalloproteinases and tissue inhibitors of metalloproteinases in newly established human oral malignant tumor lines.

We have established 11 human oral tumor lines maintained as in subcutaneous xenografts in BALB/c athymic nude mice, and examined their metastatic and invasive characteristics, and expression of type IV collagen-degrading metalloproteinases and their intrinsic inhibitors (TIMPs). These tumor lines have approximately maintained the histological appearance of the parental tumors. Generally, human tumors maintained subcutaneously in nude mice metastasize only very rarely. However, one mesenchymal tumor line, a malignant melanoma designated MTN, was found to metastasize spontaneously to the lung and a lysate of the MTN cells had a high level of type IV collagenolytic activity. Among epithelial tumor lines, SKH, derived from squamous cell carcinoma, showed high expression of TIMP-1 in Northern blotting and had low type IV collagenolytic activity. SN, derived from squamous cell carcinoma, also showed low type IV collagenolytic activity. These two squamous cell carcinoma lines showed a non-invasive growth pattern when they were implanted orthotopically into the tongues of athymic nude mice. By contrast, tumor lines which showed higher type IV collagenolytic activity had a tendency to grow invasively in mouse tongue. These findings suggest that our 11 newly established tumor lines may provide useful systems for studies of tumor biology and therapy.

Specific inhibition of epidermal growth factor receptor tyrosine kinase by 4-anilinoquinazolines.

Since the mitogenic action of EGF is mediated by ligand-induced autophosphorylation of the EGF receptor (EGFR), and EGFR is commonly overexpressed in solid human tumours, inhibitors of receptor tyrosine kinase activity (RTK) could prove to be effective antitumour agents. Screening of a compound library using an EGF-RTK enzyme prepared from human tumour derived A431 cells identified a series of potent (IC50 < 1 microM) enzyme inhibitors. These inhibitors are quinazolines bearing a variety of substituted anilines at the 4-position. The most potent 4-anilinoquinazolines (IC50 approximately equal to 20 nM) have small non-polar meta substituents on the aniline ring, and are competitive with ATP and non-competitive with substrate. The growth inhibitory activity of these agents was assessed in vitro using KB cells (human oral squamous tumour) grown in the absence or presence of EGF. A selected compound, 4-(3-chloroanilino)quinazoline (CAQ), inhibited EGF-stimulated growth in a concentration dependent manner and complete blockade was observed at concentrations (1-10 microM) which had no effect on basal growth. Selectivity of growth inhibition by CAQ was further exemplified in IGF1-stimulated KB cells where no effect was detected at concentrations which completely blocked EGF-stimulated growth. Similarly, CAQ blocked TGF alpha-stimulated growth in MCF-7 human breast cancer cells without affecting insulin-stimulated growth. These studies define a novel class of EGF-RTK inhibitors which are also potent and selective inhibitors of EGF-stimulated human tumour cell growth in vitro.

Laser blood-flow measurements in malignant tumors during photodynamic therapy (PDT) — An experimental study

Laser blood-flow measurements were made on the tumor surface of 18 human oral cancer tumors growing on the shank of nude mice during and up to 6 h after photodynamic therapy (PDT) to detect changes in microcirculation. In eight animals, the same procedure was done without PDT. Simultaneously, the skin microcirculation in the contralateral leg was measured to detect systemically caused changes in microcirculation. Two days later, specimens of the tumor region were taken and analyzed histologically. Our results suggest a mainly direct effect of PDT on the tumor cells rather than tumor necrosis caused by decreased microcirculation.

口腔腫ようにおける癌遺伝子の解析

The role of oncogenes in tumorigenesis has been observed by molecular biology and cell biology. A variety of human cancer cells from carcinomas of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, and stomach and from hematopoietic tumors and from tumors of mesenchymal origin contains mutant ras genes that are capable of transforming NIH 3 T 3 cells.In the present study five oral squamous cell cartinoma cell lines were screened for the presence of activated oncogenes by NIH 3 T 3 transfection assay coupled with the in vitro neomycin selection method. Activated c-H-ras-1 was detected in cellular DNAs from two cell lines, ZA and HOC-313, both obtained in our Department from human oral cancer tissues. HOC-313 DNA was found to lose the Msp I recognition sequence within the 11th and 12th codon of the ras protein, suggesting that HOC-313 had activation at the 12th codon, while ZA was sensitive to the enzyme digestion. To know the nature of the activation in ZA, the nucleotide sequences of exon I and exon II of c-H-ras-1 in one of the primary transfectant were determined by dideoxynucleotide chain-termination method. ZA was found to have two point mutations at the 13th and 27th codons within the first coding exon. The former change was associated with amino acid substitution from glycine to arginine but the latter was a silent mutation. The former point mutation was expected to generate a new Bgl I recognition site (GCCNNNNNGGC), which was comfirmed by the restriction analysis of ZA DNA. It is, thus, suggested that the point mutation in codon 13 of ZA c-H-ras-1 was responsible for the activation.DNAs from 33 human oral tumor tissues and from 9 human oral squamous cell carcinoma cell lines were analyzed for the loss of Msp I recognition site at the 12th codon of c-H-ras-1 using Southern blot hybridization No activation at the 12th codon was observed exceptfor HOC-313.DNAs from 19 human oral tumor tissues and from 8 human oral squamous cell carcinoma cell lines were also analyzed for the restriction fragment length polymorphism (RFLP) of the L-myc gene. Patients with only the L band (10 kb Eco RI fragment) had poor prognosis compared with those with either the S band (6 kb Eco RI fragment) or the S and L bands.

マウス移植悪性腫ように対するイミダゾール化合物の放射線増感作用について Ｖ ＰＮＣ‐１‐ＪＣＫ（ヒト上顎低分化型へん平上皮癌）に対するイミダゾール化合物の増感作用

The purpose of our investigation is to compare the radio-enhancement of three imidazole compounds to human oral malignant tumor transplanted on nude mice.Methods and evaluation: Battelle Columbus Laboratories Protocol.Tumor: PNC-1-JCK=human maxillary poorly differentiated squamous cell carcinoma.Subjects: Male CD-1 (ICR) nu/nu, BALBc nu/nu and conventional C 57 BL/6 N (age 10 weeks, 20-25g body weight).Imidazole compounds: Misonidazole (MISO) 20mg/mouse, dinitroimidazole ethanol (DNIE) 10mg/mouse and 5-methoxy carbonyl-2-methyl sulfinyl-l-methyl imidazole (KIH-3) 40mg/mouse.Tumors were transplanted to the subcutaneous region of the right thigh of CD-1 (ICR) nu/nu.Radiation: 60Co 15 Gy local radiation.These imidazole compounds were administrated into the abdomen of the CD-1 (ICR) nu/nu prior to 30 minutes of local irradiation.NK activity was compared among CD-1 (ICR) nu/nu, BALBc nu/nu and conventionalC 57 BL/6 N.Results: Radiosensitive effect was evaluated for MISO 20mg+60Co 15 Gy group. DNIE 10mg+60Co 15 Gy and KIH-3 40mg+60Co 15 Gy groups were observed to show radioprotective effects rather than radiosensitivity.% NK activity was as follows:CD-1 (ICR) nu/nu, 4.9 in YAC-1 100:1; 7.0 in RL-male 100:1. BALBc nu/nu 24.6 in YAC-1 100:1; 15.9 in RL-male 100:1. C 57 BL/6 N 21.4 in YAC-1 100:1; 24.0 in RL-male 100:1.

Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor.

Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation, and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently, Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively, and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein, and by using oligonucleotides as probes, have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore, Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells.

Sensitivity of Human Oral Tumor Cells to Human Adenovirus

Human cells in early passage cultures of benign oral tumors, normal oral tissues, normal lung tissues and, especially, in long-term established oral carcinoma cultures were highly susceptible to infection by human adenovirus types 5, 21, and 31. In contrast, replication of each adenovirus type was markedly limited in inoculated cells of newly-established oral squamous cell carcinoma cultures.