Histone deacetylase inhibitors specifically kill nonproliferating tumour cells

Conventional chemotherapeutic drugs target proliferating cells, relying on often small differences in drug sensitivity of tumour cells compared to normal tissue to deliver a therapeutic benefit. Consequently, they have significant limiting toxicities and greatly reduced efficacy against nonproliferating compared to rapidly proliferating tumour cells. This lack of selectivity and inability to kill nonproliferating cells that exist in tumours with a low mitotic index are major failings of these drugs. A relatively new class of anticancer drugs, the histone deacetylase inhibitors (HDI), are selectively cytotoxic, killing tumour and immortalized cells but normal tissue appears resistant. Treatment of tumour cells with these drugs causes both G1 phase cell cycle arrest correlated with increase p21 expression, and cell death, but even the G1 arrested cells died although the onset of death was delayed. We have extended these observations using cells that were stably arrested by either serum starvation or expression of the cyclin-dependent kinase inhibitor p16(ink4a). We report that histone deacetylase inhibitors have similar cytotoxicity towards both proliferating and arrested tumour and immortalized cells, although the onset of apoptosis is delayed by 24 h in the arrested cells. Both proliferating and arrested normal cells are unaffected by HDI treatment. Thus, the histone deacetylase inhibitors are a class of anticancer drugs that have the desirable features of being tumour-selective cytotoxic drugs that are equally effective in killing proliferating and nonproliferating tumour cells and immortalized cells. These drugs have enormous potential for the treatment of not only rapidly proliferating tumours, but tumours with a low mitotic index.

B cell chronic lymphocytic leukaemia cells have reduced capacity to upregulate expression of MHC class I in response to interferon-gamma

Aims: An important consideration in the design of a tumour vaccine is the ability of tumour-specific cytotoxic T lymphocytes (CTL) to recognise unmanipulated tumour cells in vivo. To determine whether B-CLL might use an escape strategy, the current studies compared B-CLL and normal B cell MHC class I expression. Methods: Flow cytometry, TAP allele PCR and MHC class I PCR were used. Results: While baseline expression of MHC class I did not differ, upregulation of MHC class I expression by B-CLL cells in response to IFN-gamma was reduced. No deletions or mutations of TAP 1 or 2 genes were detected. B-CLL cells upregulated TAP protein expression in response to IFN-gamma. Responsiveness of B-CLL MHC class I mRNA to IFN-gamma was not impaired. Conclusions: The data suggest that MHC class I molecules might be less stable at the cell surface in B-CLL than normal B cells, as a result of the described release of beta(2)m and beta(2)m-free class I heavy chains from the membrane. This relative MHC class I expression defect of B-CLL cells may reduce their susceptibility to CTL lysis in response to immunotherapeutic approaches.

Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine

Advanced metastatic melanoma is incurable by standard treatments, but occasionally responds to immunotherapy. Recent trials using dendritic cells (DC) as a cellular adjuvant have concentrated on defined peptides as the source of antigens, and rely on foreign proteins as a source of help to generate a cell-mediated immune response. This approach limits patient accrual, because currently defined, non-mutated epitopes are restricted by a small number of human leucocyte antigens. It also fails to take advantage of mutated epitopes peculiar to the patient's own tumour, and of CD4(+) T lymphocytes as potential effectors of anti-tumour immunity. We therefore sought to determine whether a fully autologous DC vaccine is feasible, and of therapeutic benefit. Patients with American Joint Cancer Committee stage IV melanoma were treated with a fully autologous immunotherapy consisting of monocyte-derived DC, matured after culture with irradiated tumour cells. Of 19 patients enrolled into the trial, sufficient tumour was available to make treatments for 17. Of these, 12 received a complete priming phase of six cycles of either 0.9X10(6) or 5X10(6) DC/intradermal injection, at 2-weekly intervals. Where possible, treatment continued with the lower dose at 6-weekly intervals. The remaining five patients could not complete priming, due to progressive disease. Three of the 12 patients who completed priming have durable complete responses (average duration 3 5 months +), three had partial responses, and the remaining six had progressive disease (WHO criteria). Disease regression was not correlated with dose or with the development of delayed type hypersensitivity responses to intradermal challenge with irradiated, autologous tumour. However, plasma S-100B levels prior to the commencement of treatment correlated with objective clinical response (P = 0.05) and survival (log rank P < 0.001). The treatment had minimal side-effects and was well tolerated by all patients. Mature, monocyte-derived DC preparations exposed to appropriate tumour antigen sources can be reliably produced for patients with advanced metastatic melanoma, and in a subset of those patients with lower volume disease their repeated administration results in durable complete responses.

Virus-specific CD8+ T lymphocytes within the normal human liver

The frequency and phenotype of human antiviral memory CD8(+) T cells in blood are well studied, yet little is known about their distribution within tissues. Analysis of antiviral CD8(+) T cell populations derived from a unique set of normal liver and blood samples identified a consistent population of virus-specific cells within the liver. In comparison to the circulating T cells, the liver-derived T cells were present at frequencies which were variably enriched compared to that in the blood, and showed significant differences with regard to the expression of CD45RA, CD45RO, CD95, CCR7, CD27 and CD28. The differences in these cell surface markers are consistent with a mature 'effector memory' phenotype of antigen-specific CD8(+) T cells within the liver. An enrichment of an activated subset of NKT cells (Valpha24/Vbeta11) was also observed, a finding which may be relevant to the regulation of the antiviral population:.

Overexpression of transforming growth factor-beta is associated with increased hyaluronan content and impairment of repair in Marfan syndrome aortic aneurysm

Background-Marfan syndrome (MFS), a condition caused by fibrillin-1 gene mutation is associated with aortic aneurysm that shows elastic lamellae disruption, accumulation of glycosaminoglycans, and vascular smooth muscle cell (VSMC) apoptosis with minimal inflammatory response. We examined aneurysm tissue and cultured cells for expression of transforming growth factor-beta1 to -beta3 (TGF beta 1 to 3), hyaluronan content, apoptosis, markers of cell migration, and infiltration of vascular progenitor cells (CD34). Methods and Results-MFS aortic aneurysm (6 males, 5 females; age 8 to 78 years) and normal aorta (5 males, 3 females; age 22 to 56 years) were used. Immunohistochemistry showed increased expression of TGF beta 1 to 3, hyaluronan, and CD34-positive microcapillaries in MFS aneurysm compared with control. There was increased expression of TGF beta 1 to 3 and hyaluronan in MFS cultured VSMCs, adventitial fibroblasts (AF), and skin fibroblasts (SF). Apoptosis was increased in MFS (VSMC: mean cell loss in MFS 29%, n of subjects = 5, versus control 8%, n = 3, P < 0.05; AF: 28%, n = 5 versus 7%, n = 5, P < 0.05; SF: 29%, n = 3 versus 4%, n = 3, not significant). In MFS, there was a 2-fold increase in adventitial microcapillaries containing CD34-positive cells compared with control tissue. Scratch wound assay showed absence of CD44, MT1-MMP, and beta-3 integrin at the leading edge of migration in MFS indicating altered directional migration. Western blot showed increased expression of TGF beta 1 to 3 in MFS but no change in expression of CD44, MT1-MMP, or beta-3 integrin compared with controls. Conclusions-There was overexpression of TGF-beta in MFS associated with altered hyaluronan synthesis, increased apoptosis, impaired progenitor cell recruitment, and abnormal directional migration. These factors limit tissue repair and are likely to contribute to aneurysm development.

Evaluation of oestrogen and progesterone receptor status in HER-2 positive breast carcinomas and correlation with outcome

Aim: HER-2/neu amplification occurs in 15-25% of breast carcinomas. This oncogene, also referred to as c-erbB-2, encodes a transmembrane tyrosine kinase receptor belonging to the epidermal growth factor receptor family. HER-2 over-expression is reported to be associated with a poor prognosis in breast carcinoma patients and in some studies is associated with a poorer response to anti-oestrogen therapy. These patients are less likely to benefit from CMF (cyclophosphamide, methotrexate, fluorouracil)-based chemotherapy compared with anthracycline-based chemotherapy. The aim of this study was to evaluate breast carcinomas to determine hormone receptor status and if there is a difference in breast cancer specific survival for HER-2 positive patients. Methods: A total of 591 breast carcinomas were evaluated using immunohistochemistry (IHC) for oestrogen receptor (ERp), progesterone receptor (PRp) and three different HER2 antibodies (CB11, A0485 and TAB250). Percentage of tumour cells and intensity of staining for ERp were evaluated using a semiquantitative method. Results: Of the 591 tumours, 91 (15.4%) showed 3+ membrane staining for HER-2 with one or more antibodies. Of these 91 tumours, 41 (45.1%) were ERp+/ PRp+, seven (7.7%) were ERp+/PR-, six (6.6%) were ERp-/PRp+ and 37 (40.7%) were ERp-/PR-. Of HER-2 positive tumours, 5.5% showed > 80% 3+ staining for ERp compared with 31.8% of 0-2+ HER-2 tumours; 24.2% of HER-2-positive tumours showed 60% or more cells with 2+ or 3+ staining for ERp. Treatment data were available for 209 patients and no difference was observed in breast cancer specific survival (BCSS) with HER-2 status and tamoxifen. Conclusion: Oestrogen receptor status cannot be used to select tumours for evaluation of HER-2 status, and oestrogen and progesterone receptor positivity does not preclude a positive HER-2 status. There is a higher proportion of ERp negative tumours associated with HER-2 positivity, however, more than 20% of HER-2 positive tumours show moderate or strong staining for ERp. HER-2 positive patients in this study did not show an adverse BCSS with tamoxifen treatment unlike some previous studies.

Expression of apoptotic regulatory molecules in renal cell carcinoma: Elevated expression of Fas ligand

Renal cell carcinoma (RCC) is the most common renal neoplasm. Despite being infiltrated by tumour infiltrating lymphocytes (TIL), these TIL are unable to control tumour growth in vivo, suggesting that the cytotoxic capacity of TIL against RCC is impaired, or that the tumour cells are resistant to killing and therefore escape detection by the immune system. It is postulated that the expression of apoptotic regulatory molecules in RCC favours tumour cell survival. The present study has therefore determined the expression of Fas (APO- 1/CD95), Fas ligand (Fas L) and bcl-2 in these tumours. The expression of Fas, Fas L and bcl-2 mRNA transcripts was determined in RCC, normal kidney and peripheral blood by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), following RNA extraction and cDNA synthesis from tissues and cell samples. Transcript levels were measured by densitometry after Southern blot hybridization of PCR products with internal radio-labelled oligonucleotide probes; a densitometry score was assigned to each hybridizing DNA band and expressed as a ratio of the glyceraldehyde-3-phosphate dehydrogenase content. In peripheral blood, the expression of Fas L and bcl-2 transcripts was similar between patients and normal healthy individuals; however, Fas transcript expression was significantly down-regulated in the patients' versus normal peripheral blood (P = 0.026). Most interestingly, significantly up-regulated Fas L expression was observed in RCC compared to normal kidney (P = 0.041). In contrast, bcl-2 transcripts were well represented in normal kidney but markedly decreased in RCC (P = 0.021). The expression of Fas transcripts in normal kidney and RCC was variable. These data demonstrate elevated expression of Fas L transcripts in RCC, but the functional relevance of this remains to be investigated.

Brain tumour stem cells

The dogma that the genesis of new cells is a negligible event in the adult mammalian brain has long influenced our perception and understanding of the origin and development of CNS tumours. The discovery that new neurons and glia are produced throughout life from neural stem cells provides new possibilities for the candidate cells of origin of CNS neoplasias. The emerging hypothesis is that alterations in the cellular and genetic mechanisms that control adult neurogenesis might contribute to brain tumorigenesis, thereby allowing the identification of new therapeutic strategies.

Current concepts of tumour metastasis

Background: Tumour metastasis remains the principal cause of treatment failure and poor prognosis in patients with cancer. Recent advances in our understanding of the biology of metastasis are providing novel potential targets for anti-cancer therapies. Aim: This paper reviews the current concepts in tumour metastasis. Methods: A review of Medline publications relating to the molecular biology and therapy of human tumour metastasis was conducted. Results and Discussion: Early metastasis models were based upon the premise of uninterrupted tumour growth, with the inevitable formation of distant metastases and eventual death of the patient. However, current research suggests that metastasis is an inefficient process governed by several rate-limiting steps, and that failure to negotiate these steps can lead to tumour dormancy. Successful metastatic tumour growth depends upon appropriate tumour-host microenvironment interactions and, ultimately, the development of vascularised metastases post-extravasation in the target organ. An understanding of the molecular mechanisms involved in this dynamic process will aid in the identification of therapeutic targets that may allow earlier diagnosis and more specific therapies for patients with metastasis.

Recombinant Kunjin virus replicon vaccines induce protective T-cell immunity against human papillomavirus 16 E7-expressing tumour

The persistence of the E7 oncoprotein in transformed cells in human papillomavirus (HPV)-associated cervical cancer provides a tumour-specific antigen to which immunotherapeutic strategies may be directed. Self-replicating RNA (replicon) vaccine vectors derived from the flavivirus Kunjin (KUN) have recently been reported to induce T-cell immunity. Here, we report that inclusion of a CTL epitope of HPV16 E7 protein into a polyepitope encoded by a KUN vector induced E7-directed T-cell responses and protected mice against challenge with an E7-expressing epithelial tumour. We found replicon RNA packaged into virus-like particles to be more effective than naked replicon RNA or plasmid DNA constructed to allow replicon RNA transcription in vivo. Protective immunity was induced although the E7 CTL epitope was subdominant in the context of other CTL epitopes in the polyepitope. The results demonstrate the efficacy of the KUN replicon vector system for inducing protective immunity directed towards a virally encoded human tumour-specific antigen, and for inducing multi-epitopic CTL responses. (C) 2004 Elsevier Inc. All rights reserved.

Reduction of β-Endorphin-Containing Immune Cells in Inflamed Paw Tissue Corresponds with a Reduction in Immune-Derived Antinociception: Reversible by Donor Activated Lymphocytes

The functional integrity of the immune system is essential for peripheral antinociception. Previous studies have demonstrated that immune cells elicit potent antinociception in inflamed tissues and that corticotropin-releasing factor-induced antinociception is significantly inhibited in animals that have undergone cyclosporin A (CsA)-induced immunosuppression. In this study, we examined the effect of a single bolus of CsA on inflammatory nociception. CsA-treated rats had substantially increased nociception compared with nonimmunosuppressed rats, consistent with a reduction in circulating and infiltrating lymphocytes. Furthermore, CsA-treated rats had inhibition of corticotropin-releasing factor-induced immune-derived antinociception, which was dose-dependently reversed by IV injection of concanavalin A-activated donor lymphocytes (1.0-7.0 X 10(6) cells/0.1 mL). In conclusion, our findings provided further evidence that opioid-containing immune cells are essential for peripheral analgesia. It is evident from these findings that control of inflammatory pain relies heavily on a functioning immune system.

Functional memory CD8+ T cells can be generated in vivo without evident T help

Synthetic cytotoxic T cell (CTL) epitope peptides provide an effective and safe means of vaccination against cancers and viruses, as these peptides can induce specific CD8+ effector T cells in vivo. However, the effector CD8+ T cells induced by the minimal CTL epitope peptides do not last past about 3 weeks after the induction and no functional memory CD8+ T cells are generated. It is held that simultaneous induction of CD4+ T cells by incorporating peptides containing T-helper epitopes in the vaccine at the time of primary vaccination are necessary for the induction of long-lived functional memory CD8+ T cells. We now report that, surprisingly, incorporation of medium length (>20 AA) peptides devoid of detectable T-helper epitopes in a minimal CTL epitope-based vaccine can also induce long-lasting! functional rumour antigen specific memory CD8+ T cells that are capable of promoting protection against tumour challenge. This observation may have implications for the formulation of therapeutic anti-cancer and anti-virus peptide vaccines where a strong induction of CD4 T help would be undesirable. (C) 2004 Elsevier Ltd. All rights reserved.

Tumour susceptibility to innate and adaptive immunotherapy changes during tumour maturation

Immunotherapy of tumours using T cells expanded in vitro has met with mixed clinical success suggesting that a greater understanding of tumour/T-cell interaction is required. We used a HPV16E7 oncoprotein-based mouse tumour model to study this further. In this study, we demonstrate that a HPV16E7 tumour passes through at least three stages of immune susceptibility over time. At the earliest time point, infusion of intravenous immune cells fails to control tumour growth although the same cells given subcutaneously at the tumour site are effective. In a second stage, the tumour becomes resistant to subcutaneous infusion of cells but is now susceptible to both adjuvant activated and HPV16E7-specific immune cells transferred intravenously. In the last phase, the tumour is susceptible to intravenous transfer of HPV16E7-specific cells, but not adjuvant-activated immune cells. The requirement for IFN-gamma and perforin also changes with each stage of tumour development. Our data suggest that effective adoptive T-cell therapy of tumour will need to be matched with the stage of tumour development.

Hanging-drop multicellular spheriods as a model of tumour angiogenesis

The establishment of a vascular network within tumours is a key step in the progression towards an aggressive, metastatic state, with poor prognosis. We have developed a novel in vitro model to specifically capture the interaction between endothelial cells and solid tumours. Micro-vascularised in vitro tumour constructs were produced by introducing endothelial cells to multicellular spheroids formed in hanging drops. Upon introduction, the endothelial cells migrated into the tumour spheroid, establishing tubular networks and luminal structures. This system relies on the natural pro-angiogenic capacity of multicellular spheroids, and does not require the addition of exogenous angiogenic factors, or use of extracellular-matrix substitutes.

A polytope DNA vaccine elicits multiple effector and memory CTL responses and protects against human papillomavirus 16 E7-expressing tumour

Vaccine-induced CD8 T cells directed to tumourspecific antigens are recognised as important components of protective and therapeutic immunity against tumours. Where tumour antigens have pathogenic potential or where immunogenic epitopes are lost from tumours, development of subunit vaccines consisting of multiple individual epitopes is an attractive alternative to immunising with whole tumour antigen. In the present study we investigate the efficacy of two DNA-based multiepitope('polytope') vaccines containing murine (H-2(b)) and human (HLA-A* 0201)-restricted epitopes of the E7 oncoprotein of human papillomavirus type 16, in eliciting tumour-protective cytotoxic T-lymphocyte (CTL) responses. We show that the first of these polytopes elicited powerful effector CTL responses ( measured by IFN-gamma ELISpot) and long-lived memory CTL responses ( measured by functional CTL assay and tetramers) in immunised mice. The responses could be boosted by immunisation with a recombinant vaccinia virus expressing the polytope. Responses induced by immunisation with polytope DNA alone partially protected against infection with recombinant vaccinia virus expressing the polytope. Complete protection was afforded against challenge with an E7-expressing tumour, and reduced growth of nascent tumours was observed. A second polytope differing in the exact composition and order of CTL epitopes, and lacking an inserted endoplasmic reticulum targeting sequence and T-helper epitope, induced much poorer CTL responses and failed to protect against tumour challenge. These observations indicate the validity of a DNA polytope vaccine approach to human papillomavirus E7 - associated carcinoma, and underscore the importance of design in polytope vaccine construction.