Re-expression of miR-200s in claudin-low mammary tumor cells alters cell shape and reduces proliferation and invasion potentially through modulating other miRNAs and SUZ12 regulated genes.

BACKGROUND: MicroRNAs are a class of non-coding RNAs that regulate gene expression through binding to mRNAs and preventing their translation. One family of microRNAs known as the miR-200 family is an important regulator of epithelial identity. The miR-200 family consists of five members expressed in two distinct clusters; the miR-200c/141 cluster and the miR-200b/200a/429 cluster. We have found that murine and human mammary tumor cells with claudin-low characteristics are associated with very low levels of all five miR-200s. METHODS: To determine the impact of miR-200s on claudin-low mammary tumor cells, the miR-200c/141 cluster and the miR-200b/200a/429 cluster were stably re-expressed in murine (RJ423) and human (MDA-MB-231) claudin-low mammary tumor cells. Cell proliferation and migration were assessed using BrdU incorporation and transwell migration across Matrigel coated inserts, respectively. miRNA sequencing and RNA sequencing were performed to explore miRNAs and mRNAs regulated by miR-200 re-expression while Enrichr-based pathway analysis was utilized to identify cellular functions modified by miR-200s. RESULTS: Re-expression of the miR-200s in murine and human claudin-low mammary tumor cells partially restored an epithelial cell morphology and significantly inhibited proliferation and cell invasion in vitro. miRNA sequencing and mRNA sequencing revealed that re-expression of miR-200s altered the expression of other microRNAs and genes regulated by SUZ12 providing insight into the complexity of miR-200 function. SUZ12 is a member of the polycomb repressor complex 2 that suppresses gene expression through methylating histone H3 at lysine 27. Flow cytometry confirmed that re-expression of miR-200s increased histone H3 methylation at lysine 27. CONCLUSIONS: Re-expression of miR-200s in claudin-low mammary tumor cells alters cell morphology and reduces proliferation and invasion, an effect potentially mediated by SUZ12-regulated genes and other microRNAs.

Osteopontin regulates proliferation, apoptosis, and migration of murine claudin-low mammary tumor cells.

BACKGROUND: Osteopontin is a secreted phosphoglycoprotein that is expressed by a number of normal cells as well as a variety of tumor cells. With respect to breast cancer, osteopontin has been implicated in regulating tumor cell proliferation and migration/metastasis and may serve as a prognostic indicator. However it remains unclear whether osteopontin has the same impact in all breast cancer subtypes and in particular, osteopontin's effects in claudin-low breast cancer are poorly understood. METHODS: cDNA microarrays and qRT-PCR were used to evaluate osteopontin expression in mammary tumors from MTB-IGFIR transgenic mice and cell lines derived from these tumors. siRNA was then used to determine the impact of osteopontin knockdown on proliferation, apoptosis and migration in vitro in two murine claudin-low cell lines as well as identify the receptor mediating osteopontin's physiologic effects. RESULTS: Osteopontin was expressed at high levels in mammary tumors derived from MTB-IGFIR transgenic mice compared to normal mammary tissue. Evaluation of cell lines derived from different mammary tumors revealed that mammary tumor cells with claudin-low characteristic expressed high levels of osteopontin whereas mammary tumor cells with mixed luminal and basal-like features expressed lower levels of osteopontin. Reduction of osteopontin levels using siRNA significantly reduced proliferation and migration while increasing apoptosis in the claudin-low cell lines. Osteopontin's effect appear to be mediated through a receptor containing ITGAV and not through CD44. CONCLUSIONS: Our data suggests that mammary tumors with a mixed luminal/basal-like phenotype express high levels of osteopontin however this osteopontin appears to be largely produced by non-tumor cells in the tumor microenvironment. In contrast tumor cells with claudin-low characteristics express high levels of osteopontin and a reduction of osteopontin in these cells impaired proliferation, survival and migration.

Transgenic IGF-IR overexpression induces mammary tumors with basal-like characteristics, whereas IGF-IR-independent mammary tumors express a claudin-low gene signature.

Molecular profiling has allowed a more precise classification of human cancers. With respect to breast cancer, this approach has been used to identify five subtypes; luminal A, luminal B, HER2-enriched, basal-like and claudin-low. In addition, this approach can be used to determine the type of tumor represented by particular cell lines or transgenic animal models. Therefore, this approach was utilized to classify the mammary tumors that develop in MTB-IGFIR transgenic mice. It was determined that the primary mammary tumors, which develop due to elevated expression of the type I insulin-like growth factor receptor (IGF-IR) in mammary epithelial cells, most closely resemble murine tumors with basal-like or mixed gene expression profiles and with human basal-like breast cancers. Downregulation of IGF-IR transgene in MTB-IGFIR tumor-bearing mice leads to the regression of most of the tumors, followed by tumor reappearance in some of the mice. These tumors that reappear following IGF-IR transgene downregulation do not express the IGF-IR transgene and cluster with murine mammary tumors that express a mesenchymal gene expression profile and with human claudin-low breast cancers. Therefore, IGF-IR overexpression in murine mammary epithelial cells induces mammary tumors with primarily basal-like characteristics, whereas tumors that develop following IGF-IR downregulation express a gene signature that most closely resembles human claudin-low breast tumors.

Extracellular matrix proteins and tumor angiogenesis.

Tumor development is a complex process that relies on interaction and communication between a number of cellular compartments. Much of the mass of a solid tumor is comprised of the stroma which is richly invested with extracellular matrix. Within this matrix are a host of matricellular proteins that regulate the expression and function of a myriad of proteins that regulate tumorigenic processes. One of the processes that is vital to tumor growth and progression is angiogenesis, or the formation of new blood vessels from preexisting vasculature. Within the extracellular matrix are structural proteins, a host of proteases, and resident pro- and antiangiogenic factors that control tumor angiogenesis in a tightly regulated fashion. This paper discusses the role that the extracellular matrix and ECM proteins play in the regulation of tumor angiogenesis.

Preneoplastic changes persist after IGF-IR downregulation and tumor regression.

Despite our incomplete understanding of the function of the type I insulin-like growth factor receptor (IGF-IR) in tumorigenesis, IGF-IR targeting agents have entered clinical trials for the treatment of human cancers. Previously, we have shown that downregulation of IGF-IR transgene in mammary tumors in MTB-IGFIR transengic mice results in tumor regression in a majority of the mice and most of these mice do not develop recurrent mammary tumors. In this study, we examined mammary tissue of mice that did not develop recurrent tumors. Areas of tumor regression were visible macroscopically and microscopically these lesions contained cell debris, individual cells, lipofuscin and doxycycline crystals. Three of the 12 mice also presented with considerable lobuloalveolar development. The re-expression of the IGF-IR transgene in mammary tissue with stably regressed tumors resulted in the rapid re-emergence of mammary tumors, some of which seemed to originate from the regressed mammary lesions. Thus, despite stable tumor regression after IGF-IR downregulation, mammary tissue contained preneoplastic lesions and tumors rapidly re-appear upon re-overexpression of IGF-IR transgene. Therefore, IGF-IR-targeting agents may be effective at regressing mammary tumors expressing IGF-IR, but these agents will not completely eradicate all tumor cells or restore the mammary stromal environment.

The role of dysregulated glucose metabolism in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer and also one of the most poorly understood. Other health issues that are affecting women with increasing frequency are obesity and diabetes, which are associated with dysglycemia and increased blood glucose. The Warburg Effect describes the ability of fast-growing cancer cells to preferentially metabolize glucose via anaerobic glycolysis rather than oxidative phosphorylation. Recent epidemiological studies have suggested a role for hyperglycemia in the pathogenesis of a number of cancers. If hyperglycemia contributes to tumour growth and progression, then it is intuitive that antihyperglycemic drugs may also have an important antitumour role. Preliminary reports suggest that these drugs not only reduce available plasma glucose, but also have direct effects on cancer cell viability through modification of molecular energy-sensing pathways. This review investigates the effect that hyperglycemia may have on EOC and the potential of antihyperglycemic drugs as therapeutic adjuncts.

Reversibility and recurrence of IGF-IR-induced mammary tumors.

The type-I insulin-like growth factor receptor (IGF-IR) is frequently overexpressed in breast cancer and therapeutic agents targeting IGF-IR are currently in development. The ultimate success of anti-IGF-IR therapies will depend on the extent to which established tumors remain dependent upon IGF-IR signaling for sustained growth. To investigate the potential benefits and pitfalls of targeting IGF-IR, we used a doxycycline inducible mouse model of IGF-IR initiated breast cancer. We found that downregulation of IGF-IR results in tumor-size-dependent regression to an undetectable state. Partially regressed tumors almost always resumed growth in the absence of doxycycline and a proportion of tumors that regressed to an undetectable state ultimately recurred. This re-emergence of tumor growth in the absence of doxycycline was facilitated by IGF-IR-dependent and IGF-IR-independent mechanisms. Tumor escape from IGF-IR dependence was associated with an epithelial to mesenchymal transition and upregulation of transcriptional repressors of E-cadherin. These results suggest that tumors initiated by IGF-IR have the ability to become independent of this initiating oncogene, and IGF-IR independence is associated with characteristics consistent with an epithelial to mesenchymal transition.

cAMP response element-binding protein is expressed at high levels in human ovarian adenocarcinoma and regulates ovarian tumor cell proliferation.

Approximately 90% of human ovarian tumors result from transformation of ovarian surface epithelial cells. It has been hypothesized that repeated destruction of the epithelial cells during ovulation, followed by proliferation and migration of epithelial cells to restore the ovarian surface, renders these cells susceptible to mutagenic events. One of the proteins found to promote ovarian surface epithelial cell survival and proliferation was the transcription factor, cAMP response element-binding protein (CREB). Thus, the objective of this study was to determine whether CREB was also highly expressed in tumor cells originating from the ovarian epithelium. Using an ovarian cancer tissue array, it was observed that approximately 54% of the epithelial-derived human ovarian tumors displayed moderate or high levels of CREB immunostaining, while none of the normal ovarian samples did. Comparison of CREB levels in a human ovarian tumor cell line to those of a normal ovarian epithelial cell line revealed elevated levels of CREB and phosphorylated CREB in the ovarian tumor cells. To determine whether CREB regulated proliferation and/or apoptosis in the ovarian tumor cell line, CREB expression was suppressed using RNA interference. Decreased CREB expression significantly reduced ovarian tumor cell proliferation, while there was no effect on apoptosis in these cells. Finally, we showed that CREB is highly expressed in an in vivo murine model of ovarian tumorigenesis. Therefore, CREB is frequently overexpressed in ovarian cancer where it appears to promote cell proliferation.

Transgenic overexpression of IGF-IR disrupts mammary ductal morphogenesis and induces tumor formation.

Overexpression and hyperactivation of the type I insulin-like growth factor receptor (IGF-IR) has been observed in human breast tumor biopsies. In addition, in vitro studies indicate that overexpression of IGF-IR is sufficient to transform cells such as mouse embryo fibroblasts and this receptor promotes proliferation and survival in breast cancer cell lines. To fully understand the function of the IGF-IR in tumor initiation and progression, transgenic mice containing human IGF-IR under a doxycycline-inducible MMTV promoter system were generated. Administration of 2 mg/ml doxycycline in the animals' water supply beginning at 21 days of age resulted in elevated levels of IGF-IR in mammary epithelial cells as detected by Western blotting and immunohistochemistry. Whole mount analysis of 55-day-old mouse mammary glands revealed that IGF-IR overexpression significantly impaired ductal elongation. Moreover, histological analyses revealed multiple hyperplasic lesions in the mammary glands of these 55-day-old mice. The formation of palpable mammary tumors was evident at approximately 2 months of age and was associated with increased levels of IGF-IR signaling molecules including phosphorylated Akt, Erk1/Erk2 and STAT3. Therefore, these transgenic mice provide evidence that IGF-IR overexpression is sufficient to induce mammary epithelial hyperplasia and tumor formation in vivo and provide a model to further understand the function of IGF-IR in mammary epithelial transformation.

Accelerated apoptosis in the Timp-3-deficient mammary gland.

The proapoptotic proteinase inhibitor TIMP-3 is the only molecule of this family thought to influence cell death. We examined epithelial apoptosis in TIMP-3-deficient mice during mammary gland involution. Lactation was not affected by the absence of TIMP-3, but glandular function, as measured by gland-to-body weight ratio and production of beta-casein, was suppressed earlier during post-lactational involution than in controls. Histological examination revealed accelerated lumen collapse, alveolar-epithelial loss, and adipose reconstitution in Timp-3(-/-) females. Epithelial apoptosis peaked on the first day of involution in Timp-3-null glands but at day 3 in wild-type littermates. Unscheduled activation of gelatinase-A was evident by zymography and correlated with earlier fragmentation of fibronectin in Timp-3(-/-) mammary. To obtain independent evidence of the proapoptotic effects of TIMP-3 deficiency, we introduced recombinant TIMP-3-releasing pellets into regressing Timp-3(-/-) mammary tissue and showed that this treatment rescued lumen collapse and epithelial apoptosis. Ex vivo, involuting Timp-3(-/-) mammary tissue demonstrated accelerated epithelial apoptosis that could be reduced by metalloproteinase inhibition. The physiological relevance of TIMP-3 became apparent as Timp-3(-/-) dams failed to reestablish lactation after brief cessation of suckling. Thus, TIMP-3 is a critical epithelial survival factor during mammary gland involution.

Inhibition of mammary epithelial apoptosis and sustained phosphorylation of Akt/PKB in MMTV-IGF-II transgenic mice.

IGF-II is a growth factor implicated in human cancers and animal tumor models. While the mitogenic properties of IGF-II are well documented, its ability to suppress apoptosis in vivo has never been proven. We generated independent MMTV-IGF-II transgenic mice to examine the control of epithelial apoptosis at the morphological, cellular and molecular levels during the physiological event of postlactation mammary involution. Transgenic IGF-II expression was achieved in mammary epithelium and increased IGF-II bioactivity was confirmed by phosphorylation of the insulin receptor substrate-1, a signaling molecule downstream of the type I IGF receptor. IGF-II overexpression induced a delay in mammary involution, as evident by increased mammary gland to body weight ratios and persistence of both functionally intact lobulo-alveoli and mammary epithelial cellularity. The delayed mammary involution resulted from a significant reduction in mammary epithelial apoptosis, and not from increased epithelial proliferation. Recombinant IGF-II pellets implanted into involuting mammary glands of wild-type mice provided further evidence that IGF-II protein inhibited local epithelial apoptosis. At the molecular level, phosphorylated Akt/PKB, but not Erk1 or Erk2, persisted in IGF-II overexpressors and temporally correlated with reduced epithelial apoptosis. Levels of the phosphatase PTEN were unaltered in the transgenic tissue suggesting that the maintenance of Akt/PKB phosphorylation resulted from sustained phosphorylation rather than altered dephosphorylation of PIP-3. Together, this data reveal that IGF-II inhibits apoptosis in vivo and this effect correlates with prolonged phosphorylation of Akt/PKB

The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development.

Osteoprotegerin-ligand (OPGL) is a key osteoclast differentiation/activation factor essential for bone remodeling. We report that mice lacking OPGL or its receptor RANK fail to form lobulo-alveolar mammary structures during pregnancy, resulting in death of newborns. Transplantation and OPGL-rescue experiments in opgl-/- and rank-/- pregnant females showed that OPGL acts directly on RANK-expressing mammary epithelial cells. The effects of OPGL are autonomous to epithelial cells. The mammary gland defect in female opgl-/- mice is characterized by enhanced apoptosis and failures in proliferation and PKB activation in lobulo-alveolar buds that can be reversed by recombinant OPGL treatment. These data provide a novel paradigm in mammary gland development and an evolutionary rationale for hormonal regulation and gender bias of osteoporosis in females.

Cellular turnover and extracellular matrix remodeling in female reproductive tissues: functions of metalloproteinases and their inhibitors.

Female reproductive tissues possess a unique ability to accommodate a remarkable amount of cell turnover and extracellular matrix (ECM) remodeling following puberty. Cellular structures within ovary, uterus, and mammary tissue not only change cyclically in response to ovarian hormones but also undergo differentiation during pregnancy, and eventually revert to that resembling the pre-pregnant stage. Cell proliferation, apoptosis, invasion, and differentiation are integral cellular processes that are precisely regulated in reproductive tissues, but become dysregulated in pathologies such as cancer. Explicit reorganization of ECM and basement membranes is also critical to preserve the form and function of these tissues. Here we review the evidence that coordinated spatiotemporal expression patterns of matrix metalloproteinase (MMP) genes and their tissue inhibitors (TIMPs) are important in cell and ECM turnover of the ovary, uterus, and mammary tissues. We discuss how perturbation in these gene families may impact the biology of these reproductive tissues and the factors implicated in the control of MMP and TIMP gene expression. The observed trends in MMP and TIMP expression involved in ovarian and mammary carcinomas are also presented.

Influence of the proto-oncogene c-fos on cisplatin sensitivity.

Cisplatin resistance has been associated with overexpression of the c-fos gene in a human ovarian carcinoma cell line. To determine whether the correlation between c-fos overexpression and cisplatin resistance was limited to this cell line or was a more generalized phenomenon, we investigated cisplatin sensitivity in rat fibroblast cells that overexpressed the c-fos gene. The cisplatin Ic50 values for two different c-fos transfectants, CMVc-fos and L1-3c-fos, were 7.6 +/- 0.8 and 5.6 +/- 1.0 microM, respectively, whereas the cisplatin Ic50 value for the parental line, 208F, was 2.4 +/- 0.1 microM. This represented a 3.2- and 2.3-fold resistance to cisplatin for CMVc-fos and L1-3c-fos cells, respectively. The correlation between c-fos expression and cisplatin resistance also was examined in a human ovarian carcinoma cell line, 2008, and its cisplatin-resistant variant, C13*. Expression of c-fos was elevated slightly at both the mRNA and protein levels in the C13* cells compared with 2008 cells, and c-Fos protein levels were induced in C13* cells following cisplatin treatment. In addition, it was observed that C13* cells were significantly more sensitive than 2008 cells to a c-fos antisense oligonucleotide. The Ic50 values for the c-fos antisense oligonucleotide were 19.9 +/- 5.0 pmol for C13* cells and 58.1 +/- 6.0 pmol for 2008 cells (P = 0.0012). Furthermore, combinations of c-fos antisense and cisplatin reduced the amount of cisplatin required to kill 50% of the C13* cells, although the interaction was not synergistic. These results suggest that expression of the c-fos gene can influence cisplatin sensitivity, and that c-fos antisense oligonucleotide based therapy may be effective at killing parental and cisplatin-resistant ovarian carcinoma cells, either alone or in combination with cisplatin.

Mitochondrial membrane potential regulation is independent of c-fos expression.

Tumour cells contain mitochondria with elevated membrane potentials compared with normal cells, and thus this feature provides a selective target for destroying tumour cells. To improve mitochondrial-based therapies, a better understanding of the factors involved in regulating mitochondria are required. Since v-fos overexpression has been shown to elevate mitochondrial membrane potentials in rat fibroblasts, we investigated whether the human homologue, c-fos, was also capable of regulating the mitochondrial membrane potential in cells. Rat fibroblasts transfected with the c-fos gene did not accumulate more rhodamine 123 (Rh123) nor did they retain this Rh123 for extended periods of time compared with their parental line. Moreover, there was no difference in survival following dequalinium chloride (Deca) treatment between transfectants and controls. Similarly, reduction of c-fos expression in rat fibroblasts did not significantly alter their mitochondrial membrane potential. In addition, human ovarian carcinoma cells, which overexpress the c-fos gene, did not accumulate more Rh123 nor were they hypersensitive to Deca compared with their parental line. In another human ovarian carcinoma cell line, selection of variants with lower mitochondrial membrane potential did not alter c-fos mRNA or protein levels. These data suggest that alterations in c-fos expression do not regulate the magnitude of the mitochondrial membrane potential.

Timp-1 is important for epithelial proliferation and branching morphogenesis during mouse mammary development.

The dynamic process of mammary ductal morphogenesis depends on regulated epithelial proliferation and extracellular matrix (ECM) turnover. Epithelial cell-matrix contact closely dictates epithelial proliferation, differentiation, and survival. Despite the fact that tissue inhibitors of metalloproteinases (Timps) regulate ECM turnover, their function in mammary morphogenesis is unknown. We have delineated the spatiotemporal expression of all Timps (Timp-1 to Timp-4) during discrete phases of murine mammary development. Timp mRNAs were abundant in mammary tissue, each displaying differential expression patterns with predominant localization in luminal epithelial cells. Timp-1 mRNA was unique in that its expression was limited to the stage at which epithelial proliferation was high. To assess whether Timp-1 promotes or inhibits epithelial cell proliferation we manipulated mammary Timp-1 levels, genetically and biochemically. Down-regulation of epithelial-derived Timp-1 in transgenic mice, by mouse mammary tumor virus promoter-directed Timp-1 antisense RNA expression, led to augmented ductal expansion and increased number of ducts (P < 0.004). In these transgenics the integrity of basement membrane surrounding epithelial ducts, as visualized by laminin-specific immunostaining, was breached. In contrast to these mice, ductal expansion was markedly attenuated in the proximity of implanted recombinant Timp-1-releasing pellets (rTIMP-1), without an increase in basement membrane deposition around migrating terminal end buds. Epithelial proliferation and apoptosis were measured to determine the basis of altered ductal expansion. Luminal epithelial proliferation was increased by 55% (P < 0.02) in Timp-1-reduced transgenic mammary tissue and, conversely, decreased by 38% (P < 0.02) in terminal end buds by implanted rTIMP-1. Epithelial apoptosis was minimal and remained unaffected by Timp-1 manipulations. We conclude that Timps have an integral function in mammary morphogenesis and that Timp-1 regulates mammary epithelial proliferation in vivo, at least in part by maintaining basement membrane integrity.

Nucleotide excision repair in the human ovarian carcinoma cell line (2008) and its cisplatin-resistant variant (C13*).

Repair of cisplatin-damaged DNA was investigated in a human ovarian carcinoma cell line (2008) and its cisplatin-resistant variant (C13*) using a host-cell reactivation (HCR) assay. The HCR of cisplatin-damaged adenovirus (Ad) was not significantly different in C13* cells compared to 2008 cells. The cisplatin concentrations required to reduce the amount of viral DNA replicated to 50% were 0.12 +/- 0.02 microM and 0.10 +/- 0.01 microM after 48 h of repair in 2008 and C13* cells respectively. Similarly, the cisplatin concentration required to reduce the expression of a reporter gene inserted in the viral DNA was not significantly altered in C13* cells compared to the parental line (IC50 values were 0.28 +/- 0.04 microM in 2008 cells and 0.17 +/- 0.06 microM in C13* cells after 48 h of repair). Pretreatment of the cells with cisplatin, immediately prior to Ad infection, did not significantly alter the HCR of cisplatin-damaged Ad in either cell type. In addition, a cisplatin-sensitive variant derived from the C13* cells, namely the RH4 cells, did not differ significantly from either the 2008 or C13* cells in their ability to reactivate cisplatin-damaged Ad. Furthermore, a component of the nucleotide excision repair (NER) pathway, DNA polymerase alpha, was investigated using the competitive inhibitor aphidicolin. The combination of cisplatin and aphidicolin resulted in similar synergistic growth inhibition in both the 2008 and C13* cells providing additional support to the HCR results which suggest that enhanced NER is not responsible for the cisplatin resistance in C13* cells.

Cross-resistance to cisplatin in cells resistant to photofrin-mediated photodynamic therapy.

This study shows that a Photofrin-induced photodynamic therapy-resistant variant (RIF-8A) of a radiation-induced fibrosarcoma-1 cell line (RIF-1) is cross-resistant to cis-diamminedichloroplatinum(II) (cisplatin). This is the first study to show cross-resistance to cisplatin in photodynamic therapy-resistant variants in vitro. Resistance does not appear to be the result of elevated glutathione levels since neither the resistant variant (RIF-8A) nor the parental line (RIF-1) varied in total glutathione levels. However, cisplatin-DNA adduct levels differed significantly between the two cell types. Immediately following a 1-h exposure to cisplatin (50 microM), RIF-1 cells contained 44.6 +/- 2.0 (SEM) pg platinum/micrograms DNA while RIF-8A cells contained 24.8 +/- 6.3 pg platinum/micrograms DNA. In addition, the resistant variant had decreased plasma and mitochondrial membrane potentials. The plasma and mitochondrial membranes of the resistant variant accumulated 3- and 3.6-fold less rhodamine 123, respectively. The difference in rhodamine 123 accumulation could not be attributed to elevated P-glycoprotein expression because both the parental line and the variant contained similar amounts of P-glycoprotein. In conclusion, alterations in the plasma and/or mitochondrial membrane potentials may provide cells with a survival advantage when challenged with either photodynamic therapy or cisplatin in vitro. This appears to be a novel mechanism of resistance.