Cytogenetic evidence of the multistep origin of head and neck squamous cell carcinomas.

BACKGROUND: Head and neck squamous cell carcinomas are associated with tobacco and alcohol use; therefore, the incidence of this type of tumor is expected to rise in the future as a result of the increasing numbers of female and adolescent smokers. Previous reports of cytogenetic analysis of this type of tumor have implicated a number of chromosomal regions in recurring changes, but no clear pattern of characteristic changes has emerged. PURPOSE: We have undertaken cytogenetic analysis of 10 cell lines which were established from squamous cell carcinomas of the head and neck, to determine the possible sites of additional tumor suppressor genes and oncogenes that may contribute to malignant transformation. METHODS: Metaphases were harvested from cultures of cells in the exponential growth phase, following exposure to Colcemid (demecolcine) at a final concentration of 30 ng/mL for 5 hours. Air-dried slides were G-banded using trypsin and Giemsa. Fifteen metaphases were photographed and fully karyotyped. RESULTS: We observed that several chromosomal regions were lost at high frequency, including 18q (10 of 10 lines), 10p (eight of 10 lines), 3p (six of 10 lines), 8p (seven of 10 lines), and the short arms of the acrocentric chromosomes (seven of 10 lines). Nine of 10 lines had additional copies of 7p. We also noted clustering of breakpoints in a number of chromosome bands, including 1p22, 10q11.2, 11q13, and the short arms of the acrocentric chromosomes. CONCLUSION: The observation of loss of multiple chromosomal regions in a significant number of lines analyzed is consistent with the theory that tumorigenesis occurs as the result of the accumulation of a number of genetic alterations, as proposed for colorectal carcinoma. The high frequency with which these changes are seen suggests that genes located in these regions have a role in the etiology of this type of tumor.

Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultured from human squamous cell carcinomas.

We have established cell lines from six human squamous cell carcinomas (SCC) of the epidermis and tongue, using culture methods previously developed for clonal growth and serial cultivation of normal keratinocytes. The SCC lines all form rapidly growing, well-differentiated SCC's or progressively growing squamous cysts in nude mice. In contrast to normal keratinocytes, SCC cells form unstratified or very poorly stratifying colonies and do not require epidermal growth factor for sustained growth. The SCC lines vary in their requirement for a fibroblast feeder layer to support clonal growth, as normal keratinocytes possess. Only one line forms large, progressively growing colonies at high efficiency in semisolid medium; the other five lines exhibit only a small amount of abortive growth in semisolid medium, after which the cells appear to rapidly degenerate. These results demonstrate that SCC's often grow as established lines in culture, but they frequently possess in vitro growth requirements similar to those of normal keratinocytes. Consequently, neither semisolid medium nor standard surface culture media are appropriate for initiating primary SCC cultures or for selecting transformants out of carcinogen-treated keratinocyte populations, because they do not provide conditions permissive for the growth of many malignant keratinocytes.

Chromosome changes characterizing in vitro response to radiation in human squamous cell carcinoma lines.

We have analyzed the karyotypes of a series of 16 cell lines derived from human squamous cell carcinomas of the head and neck. When the cell lines were grouped according to their in vitro response to radiation, it was observed that some recurrent chromosomal changes occurred with differing frequencies between groups. Radiation resistance was associated with clusters of breakpoints in bands 1p22, 3p21, and 8p11.2 and deletion of distal 14q, while relative radiation sensitivity was associated with a high frequency of breakpoints in 11q13 and duplication of distal 14q. The regions identified point to the possible locations of genes involved in the response to radiation and could provide a series of markers with which to predict response to radiation therapy.

Synergistic cytotoxicity and DNA strand break formation by bromodeoxyuridine and bleomycin in human tumor cells.

5-Bromo-2'-deoxyuridine (BrdUrd) is a thymidine analogue whose cellular effects are related to its incorporation into DNA. BrdUrd is a known radiosensitizing agent that could potentially enhance the activity of chemotherapeutic agents that interact directly with DNA. Therefore we studied the interaction of BrdUrd and bleomycin in a human head and neck squamous carcinoma cell line, SQ20B. Using a colony-forming assay and analyzing results by the median-effect method, we have shown that there is synergistic cytotoxicity between BrdUrd and bleomycin. Synergism is evident when BrdUrd is administered prior to bleomycin or when the two drugs are applied simultaneously and is evident at a variety of BrdUrd:bleomycin concentration ratios. Alkaline elution of DNA from cells exposed to BrdUrd and bleomycin demonstrated greater single strand break formation than expected from the individual single strand break frequencies induced by each drug alone. BrdUrd did not affect the rate of repair of bleomycin-induced single strand breaks or the formation of double strand breaks. Although the mechanism of this interaction at the molecular level is unclear, our studies suggest that a direct interaction of bleomycin with BrdUrd-substituted DNA may be the cause of the synergism of these two agents.

p53 gene mutations and abnormal retinoblastoma protein in radiation-induced human sarcomas.

The potentially carcinogenic effect of therapeutic irradiation has been recognized for many years. Second malignancies, usually sarcomas, are known to arise within or at the edge of radiation fields after a period of several years after the initial radiation exposure. We analyzed tumor cells derived from seven radiation-induced tumors for abnormalities in tumor suppressor genes p53 and retinoblastoma at the DNA sequence and/or protein level. p53 mutations were detected by exon-specific polymerase chain reaction amplification and single-strand conformation polymorphism analysis of exons 5-8 followed by direct genomic sequencing of those tumors exhibiting a variant pattern. The p53 gene was abnormal in three of six sarcomas studied. Retinoblastoma gene analysis was performed by Western immunoblot; retinoblastoma protein was under-phosphorylated in three of seven tumors and absent in one other. In all, six of seven radiation-induced human tumors have abnormalities of one or both suppressor genes. Inactivation of tumor suppressor genes by ionizing radiation may contribute to radiation carcinogenesis.

Genetic radiotherapy overcomes tumor resistance to cytotoxic agents.

We report that radiation enhances gene therapy of a radioresistant tumor by upregulating the induction of a chimeric gene encoding a radiosensitizing protein, tumor necrosis factor alpha (TNF-alpha). We ligated the radiation-inducible CArG elements of the radiation-inducible Egr-1 promoter/enhancer region upstream to the transcriptional start site of the human TNF cDNA (pE425-TNF). This construct was transfected using cationic liposomes into the variant murine fibrosarcoma cell line, P4L. The P4L cell line was both radioresistant (D0 = 188) and resistant to TNF. After a single intratumoral injection of 10 micrograms of pE425-TNF in cationic liposomes and two 20-Gy doses of irradiation, mean tumor volumes were significantly reduced in P4L tumors as compared to those receiving either pE425-TNF in liposomes or radiation alone (P = 0.01). TNf protein in P4L tumors was induced by radiation as high as 29 times control levels and remained detectable for 14 days. Our data indicate that combined gene therapy using liposomes, together with ionizing radiation to locally activate the induction of a radiosensitizing protein, is successful at overcoming resistance to both TNF and radiation.

Footprinting of individual tumors and their variants by constitutive cytokine expression patterns.

We have examined the cytokine mRNA expression profile of six different human cell lines derived from Ewing sarcomas using polymerase chain reaction and found each to constitutively express a characteristic pattern. Furthermore, each cell line differed in the levels of secreted cytokines. We also analyzed the expression of several cytokines in murine UV-induced sarcomas and their heritably stable progressive variants. Each murine tumor also constitutively expressed a large number of cytokines, and in some cases, the more malignant variants differed from their parental tumors. These results demonstrate that tumors of the same type, and even in the same lineage, can have distinct cytokine expression and/or secretion profiles. Some cytokines may stimulate tumor growth while others may have antitumor effects. Cytokine therapy may be tailored depending upon the cytokine profile of the individual malignancy.

Loss of ceramide production confers resistance to radiation-induced apoptosis.

Ionizing radiation mediates cell death, in part, through chromosomal damage following one or more cell divisions. X-rays also induce programmed cell death (apoptosis) in some cell types both in vitro and in vivo. Both neutral and acidic sphingomyelinases, which generate the lipid second messenger ceramide, are reported to induce apoptosis following ionizing radiation and other death signals such as tumor necrosis factor alpha and Fas ligand. Herein we report that a loss of ceramide production from a neutral sphingomyelinase generates a radioresistant phenotype as measured by a marked decrease in apoptosis. A WEHI-231 subline made deficient in ceramide production was found to be resistant to apoptosis compared with the parental subline following treatment with X-rays. The resistant subline underwent two to three subsequent cell divisions following X-irradiation, confirming that X-rays induce cell death through both mitotic and apoptotic mechanisms. These data suggest that loss of ceramide production following X-rays represents an extranuclear mechanism for the development of radioresistance. Modulation of extranuclear signals may increase tumor cell killing following radiation and represent new cellular targets for cancer therapy.

Increased glutathione peroxidase activity in a human sarcoma cell line with inherent doxorubicin resistance.

Several mechanisms of drug resistance have been defined using cell lines selected for resistance in vitro. However, the relevance of these to tumor cell resistance in vivo remains unclear. We established tumor cell lines from biopsies of human sarcomas before and after doxorubicin therapy. One pretreatment sarcoma line, STSAR90, was 6-fold less sensitive to doxorubicin than was a normal fibroblast line, AG1522. The sensitivities of six other sarcoma lines were similar to that of AG1522. STSAR90 cells did not overexpress P-glycoprotein mRNA, by Northern analysis with the pCHP1 complementary DNA fragment. Photoaffinity labeling with the vinblastine analogue N-(p-azido-3-125I-salicyl)-N'-beta-aminoethylvindesine did not show increased P-glycoprotein concentrations. Accumulation of [3H]daunomycin was not decreased in STSAR90 compared with a less resistant sarcoma line, STSAR11, nor was the doxorubicin sensitivity of STSAR90 increased by coincubation with verapamil. Glutathione levels were twice as high in STSAR90 as in STSAR11, and glutathione peroxidase activity was 3.5- to 6-fold higher. This was due mostly to an increase in selenium-dependent peroxidase activity. After exposure to doxorubicin, STSAR90 cells formed only half as much measurable hydroxyl radical as STSAR11, as detected by electron spin resonance spectrometry. Doxorubicin sensitivity was increased in STSAR90 cells when intracellular glutathione levels were reduced by buthionine sulfoximine. These results indicate that multidrug resistance due to P-glycoprotein-mediated drug efflux is not the only mechanism of doxorubicin resistance that occurs in sarcomas and that glutathione peroxidase-dependent detoxification of doxorubicin-induced oxygen radicals may contribute to clinical doxorubicin resistance.

Gene therapy targeted by radiation preferentially radiosensitizes tumor cells.

Transcriptional regulation of the promoter/enhancer region of the Egr-1 gene is activated by ionizing radiation. We linked DNA sequences from the promotor region of Egr-1 to a complementary DNA sequence which encodes human tumor necrosis factor (TNF) alpha, a radiosensitizing cytokine. The Egr-TNF construct was transfected into a human cell line of hematopoietic origin, HL525, which was used in an experimental animal system. HL525 (clone 2) cells containing the Egr-TNF construct which exhibits radiation induction of TNF-alpha were injected into human xenografts of the radioresistant human squamous cell carcinoma cell line SQ-20B. Animals treated with radiation and clone 2 demonstrated an increase in tumor cures compared with animals treated with radiation alone or unirradiated animals given injections of clone 2 alone. No increase in local or systemic toxicity was observed in the combined treatment group. The combination of gene therapy and radiation therapy enhances tumor cures without increasing normal tissue toxicity and is a new paradigm for cancer treatment.

Decreasing the apoptotic threshold of tumor cells through protein kinase C inhibition and sphingomyelinase activation increases tumor killing by ionizing radiation.

Approximately 30% of cancer deaths result from the failure to control local and regional tumors. The goal of radiotherapy is to maximize local and regional tumor cell killing while minimizing normal tissue destruction. Attempts to enhance radiation-mediated tumor cell killing using halogenated pyrimidines, antimetabolites, and other DNA-damaging agents or sensitizers of hypoxic tumor cells have met with only modest clinical success. In an unique strategy to modify tumor radiosensitivity, we used an inhibitor of the protein kinase C group A and B isoforms, chelerythrine chloride (chelerythrine), to enhance the killing effects of ionizing radiation (IR). Protein kinase C activity plays a central role in cellular proliferation, differentiation, and apoptosis. Chelerythrine increases sphingomyelinase activity and enhances IR-mediated cell killing through induction of apoptotic tumor cell death in a radioresistant tumor model both in vitro and in vivo. Although previous reports have suggested that IR-mediated apoptosis correlates with tumor volume reduction, we demonstrate for the first time that lowering the apoptotic threshold increases tumor cell killing in vivo.

Modulation of apoptotic response of a radiation-resistant human carcinoma by Pseudomonas exotoxin-chimeric protein.

Strategies to sensitize human tumors that are resistant to apoptosis have been clinically unsuccessful. We demonstrate that a structurally modified chimeric Pseudomonas exotoxin, PEdelta53L/TGF-alpha/KDEL, with binding specificity for the epidermal growth factor receptor, markedly enhances sensitivity of human xenografts to radiation killing. Exposure to PEdelta53L/TGF-alpha/KDEL decreases the apoptotic threshold through protein synthesis inhibition and simultaneous production of ceramide in tumor cells that lack functional p53 protein. In contrast, no increase in local or systemic toxicity was observed with the chimeric toxin and radiation. We conclude that biochemical targeting of the chimeric toxin and physical targeting of ionizing radiation may increase the therapeutic ratio in the treatment of human cancers with alterations of p53 expression. This strategy offers a high therapeutic potential for Pseudomonas exotoxin A chimeric proteins and irradiation.

X-ray and cis-diamminedichloroplatinum(II) cross-resistance in human tumor cell lines.

Clinical studies have suggested a close correlation between cis-diamminedichloroplatinum(II) (cisplatin) and radiation resistance. To determine whether this cross-resistance is due to an inherent cellular resistance to both agents, ten early passage human tumor cell lines were examined for their radiation and cisplatin sensitivity in vitro. Previous studies have suggested that these early passage tumor cell lines retain many of their in vivo characteristics and are therefore good models for tumor cells in vivo. Radioresistance was strongly associated with cisplatin resistance in these cell lines. Four of the cell lines examined were radioresistant, having Dos greater than 2.0 Gy. These four lines were also resistant to cisplatin, with the dose reducing survival to 10% greater than 1.29 microM. The remaining six cell lines had Dos ranging from 1.07 to 1.57 Gy of X-ray and doses reducing survival to 10% of less than 0.83 microM cisplatin. Because early passage human tumor cell lines were used, resistance or sensitivity to radiation and cisplatin most likely developed in vivo and was not due to selection in vitro. These results indicate that cross-resistance between cisplatin and radiation in vivo is probably due primarily to an inherent cellular resistance to these agents and not necessarily to the tumor microenvironment in situ.

Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation.

The family of vascular endothelial growth factor (VEGF) proteins include potent and specific mitogens for vascular endothelial cells that function in the lation of angiogenesis Inhibition of VEGF-induced angiogenesis either by neutralizing antibodies or dominant-negative soluble receptor, blocks the growth of primary and metastatic experimental tumors Here we report that VEGF expression is induced in Lewis lung carcinomas (LLCs) both in vitro and vivo after exposure to ionizing radiation (IR) and in human tumor cell lines (Seg-1 esophageal adenocarcinoma, SQ20B squamous cell carcinoma, T98 and U87 glioblastomas, and U1 melanoma) in vitro. The biological significance of IR-induced VEGF production is supported by our finding that treatment of tumor-bearing mice (LLC, Seg-1, SQ20B, and U87) with a neutralizing antibody to VEGF-165 before irradiation is associated with a greater than additive antitumor effect. In vitro, the addition of VEGF decreases IR-induced killing of human umbilical vein endothelial cells, and the anti-VEGF treatment potentiates IR-induced lethality of human umbilical vein endothelial cells. Neither recombinant VEGF protein nor neutralizing antibody to VEGF affects the radiosensitivity of tumor cells These findings support a model in which induction of VEGF by IR contributes to the protection of tumor blood vessels from radiation-mediated cytotoxicity and thereby to tumor radioresistance.

Potentiation of the antitumor effect of ionizing radiation by brief concomitant exposures to angiostatin.

Angiostatin, a proteolytic fragment of plasminogen, inhibits the growth of primary and metastatic tumors by suppressing angiogenesis. When used in combination with ionizing radiation (IR), angiostatin demonstrates potent antitumor synergism, largely caused by inhibition of the tumor microvasculature. We report here the temporal interaction of angiostatin and IR in Lewis lung carcinoma (LLC) tumors growing in the hind limbs of syngeneic mice. Tumors with an initial mean volume of 510 +/- 151 mm3 were treated with IR alone (20 Gy x 2 doses on days 0 and 1), angiostatin alone (25 mg/kg/day divided twice daily) on days 0 through 13, or a combination of the two as follows: (a) IR plus angiostatin (days 0 through 13); (b) IR plus angiostatin (days 0 and 1); and (c) IR followed by angiostatin beginning on the day after IR completion and given daily thereafter (days 2 through 13). By day 14, tumors in untreated control mice had grown to 6110 +/- 582 mm3, whereas in mice treated with: (a) IR alone, tumors had grown to 2854 +/- 338 mm3 (P < 0.05 compared with untreated controls); and (b) angiostatin alone, tumors had grown to 3666 +/- 453 mm3 (P < 0.05 compared with untreated controls). In combined-treatment groups, in mice treated with: (a) IR plus longer-course angiostatin, tumors reached 2022 +/- 282 mm3 (P = 0.036 compared with IR alone); (b) IR followed by angiostatin, tumors reached 2677 +/- 469 mm3 (P > 0.05 compared with IR alone); and (c) IR plus short-course angiostatin, tumors reached 1032 +/- 78 mm3 (P < 0.001 compared with IR alone). These findings demonstrate that the efficacy of experimental radiation therapy is potentiated by brief concomitant exposure of the tumor vasculature to angiostatin.

NM-3, an isocoumarin, increases the antitumor effects of radiotherapy without toxicity.

We examined the effects of a new antiangiogenic isocoumarin, NM-3, as a radiation modifier in vitro and in vivo. The present studies demonstrate that NM-3 is cytotoxic to human umbilical vein endothelial cells (HUVECs) but not to Lewis lung carcinoma (LLC) cells nor Seg-1, esophageal adenocarcinoma cells, in clonogenic survival assays. When HUVEC cultures are treated with NM-3 combined with ionizing radiation (IR), additive cytotoxicity is observed. In addition, the combination of NM-3 and IR inhibits HUVEC migration to a greater extent than either treatment alone. The effects of treatment with NM-3 and IR were also evaluated in tumor model systems. C57BL/6 female mice bearing LLC tumors were given injections for 4 consecutive days with NM-3 (25 mg/kg/day) and treated with IR (20 Gy) for 2 consecutive days. Combined treatment with NM-3 and IR significantly reduced mean tumor volume compared with either treatment alone. An increase in local tumor control was also observed in LLC tumors in mice receiving NM-3/IR therapy. When athymic nude mice bearing Seg-1 tumor xenografts were treated with NM-3 (100 mg/kg/day for 4 days) and 20 Gy (four 5 Gy fractions), significant tumor regression was observed after combined treatment (NM-3 and IR) compared with IR alone. Importantly, no increase in systemic or local tissue toxicity was observed after combined treatment (NM-3 and IR) when compared with IR alone. The bioavailability and nontoxic profile of NM-3 suggests that the efficacy of this agent should be tested in clinical radiotherapy.

Basal Tumor Cell Isolation and Patient-Derived Xenograft Engraftment Identify High-Risk Clinical Bladder Cancers.

Strategies to identify tumors at highest risk for treatment failure are currently under investigation for patients with bladder cancer. We demonstrate that flow cytometric detection of poorly differentiated basal tumor cells (BTCs), as defined by the co-expression of CD90, CD44 and CD49f, directly from patients with early stage tumors (T1-T2 and N0) and patient-derived xenograft (PDX) engraftment in locally advanced tumors (T3-T4 or N+) predict poor prognosis in patients with bladder cancer. Comparative transcriptomic analysis of bladder tumor cells isolated from PDXs indicates unique patterns of gene expression during bladder tumor cell differentiation. We found cell division cycle 25C (CDC25C) overexpression in poorly differentiated BTCs and determined that CDC25C expression predicts adverse survival independent of standard clinical and pathologic features in bladder cancer patients. Taken together, our findings support the utility of BTCs and bladder cancer PDX models in the discovery of novel molecular targets and predictive biomarkers for personalizing oncology care for patients.

Molecular targets to overcome radioresistance.

Our laboratory has cultured cells from 23 patients with head and neck carcinoma and 13 patients with soft tissue sarcoma. The data suggest that, in some patients, inherent tumor cell radioresistance contributes to radiotherapy failure. This paper examines possible mechanisms that lead to increased cell survival following radiation exposure, and their potential as targets to overcome the radioresistant patient population.

The interaction between recombinant human tumor necrosis factor and radiation in 13 human tumor cell lines.

Tumor necrosis factor (TNF) was cytotoxic at concentrations of 10 to 1000 units/ml to 12 of 14 human tumor cell lines. Synergistic or additive cell killing between TNF and radiation was observed in 7 of 10 tumor cell lines, while independent tumor cell killing by each agent occurred in two tumor cell lines. The maximum synergistic effect was observed when TNF was added 4-12 hr prior to irradiation. This interaction was absent when TNF was added after irradiation. TNF also reduced potentially lethal damage repair in 3 of 5 cell lines tested. Possible mechanisms of interaction of TNF and X rays including induction of hydroxyl radicals and subsequent DNA damage by TNF and radiation are discussed.

An important step in radiation carcinogenesis may be inactivation of cellular genes.

The loss of genetic material may result in a predisposition to malignant disease. The best studied example is retinoblastoma where deletion or transcriptional inactivation of a specific gene is associated with the development of the tumor. When hereditary retinoblastoma patients are treated with radiation, the incidence of osteosarcoma within the treatment field is extremely high compared to other cancer patients treated with radiotherapy. These data, together with cytogenetic and molecular data on the development of acute non-lymphocytic leukemia secondary to radiotherapy and chemotherapy treatment suggest that radiation-induced deletions of critical DNA sequences may be an important event in radiation carcinogenesis. Therefore, we propose that radiation-induced tumors may result from deletion of tissue specific regulatory genes. Base alterations caused by radiation in dominantly transforming oncogenes may also contribute to radiation carcinogenesis.