Harm Reduction: A Missing Piece to the Holistic Care of Patients Who Inject Drugs.

Background: The rise in injection drug use (IDU) has led to an increase in drug-related infections. Harm reduction is an important strategy for preventing infections among people who inject drugs (PWID). We attempted to evaluate the harm reduction counseling that infectious diseases physicians provide to PWID presenting with infections. Methods: An electronic survey was distributed to physician members of the Emerging Infections Network to inquire about practices used when caring for patients with IDU-related infections. Results: In total, 534 ID physicians responded to the survey. Of those, 375 (70%) reported routinely caring for PWID. Most respondents report screening for human immunodeficiency virus (HIV) and viral hepatitis (98%) and discussing the risk of these infections (87%); 63% prescribe immunization against viral hepatitis, and 45% discuss HIV preexposure prophylaxis (PrEP). However, 55% of respondents (n = 205) reported not counseling patients on safer injection strategies. Common reasons for not counseling included limited time and a desire to emphasize antibiotic therapy/medical issues (62%), lack of training (55%), and believing that it would be better addressed by other services (47%). Among respondents who reported counseling PWID, most recommended abstinence from IDU (72%), handwashing and skin cleansing before injection (62%), and safe disposal of needles/drug equipment used before admission (54%). Conclusions: Almost all ID physicians report screening PWID for HIV and viral hepatitis and discussing the risks of these infections. Despite frequently encountering PWID, fewer than half of ID physicians provide safer injection advice. Opportunities exist to standardize harm reduction education, emphasizing safer injection practices in conjunction with other strategies to prevent infections (eg, HIV PrEP or hepatitis A virus/hepatitis B virus vaccination).

Potential cost savings associated with dose rounding antineoplastic monoclonal agents.

PURPOSE: This study explored the potential financial benefits associated with dose rounding three costly cancer agents: bevacizumab, trastuzumab, and cetuximab. METHODS: Electronic chemotherapy health record software was queried to identify inpatient and outpatient use of bevacizumab, trastuzumab, and cetuximab. Available drug vial sizes were noted. Costs of actual doses prescribed were compared to theoretically reduced doses (5% and 10%) adjusted to the nearest vial size. Only doses resulting in a decrease in the number of vials qualified for dose rounding. New doses were analyzed for potential cost savings considering the percent-change from the original dose. All institutional review board procedures were followed. RESULTS: In all, 425 doses of bevacizumab, trastuzumab, and cetuximab were identified. At a 5% dose reduction, 51 doses (12%) qualified for dose rounding, translating to a potential cost savings of $60,648 ($6,188, $52,640, and $1,820, respectively). Although a 5% limit was set, the average change in dose did not exceed 2.5%. At a 10% dose reduction, 124 doses (29%) qualified for dose rounding, translating to a potential cost savings of $112,585 ($26,520, $80,605, and $5,460, respectively). With the 10% dose reduction, the average change in dose did not surpass 6.1%. Projected annual savings were calculated as $181,944 or $337,755, depending on the rounding limit. CONCLUSION: Consultation with key physicians regarding the proposed percent reduction resulted in a 10% dose reduction for all cases when utilizing these three agents. Implementation of a dose rounding protocol for bevacizumab, trastuzumab, and cetuximab represents a potentially substantial cost savings at this institution.

A retinoblastoma allele that is mutated at its common E2F interaction site inhibits cell proliferation in gene-targeted mice.

The retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1(ΔG)) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1(ΔG/ΔG) mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1(ΔG/ΔG) MEFs display a magnitude of E2F target gene derepression similar to that of Rb1(-/-) cells, even though Rb1(ΔG/ΔG) cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1(ΔG/ΔG) MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G1 to arrest proliferation. Some Rb1(ΔG/ΔG) mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non-E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified.

SNS01-T modulation of eIF5A inhibits B-cell cancer progression and synergizes with bortezomib and lenalidomide.

The high rates of recurrence and low median survival in many B-cell cancers highlight a need for new targeted therapeutic modalities. In dividing cells, eukaryotic translation initiation factor 5A (eIF5A) is hypusinated and involved in regulation of protein synthesis and proliferation, whereas the non-hypusinated form of eIF5A is a potent inducer of cell death in malignant cells. Here, we demonstrate the potential of modulating eIF5A expression as a novel approach to treating B-cell cancers. SNS01-T is a nonviral polyethylenimine-based nanoparticle, designed to induce apoptosis selectively in B-cell cancers by small interfering RNA-mediated suppression of hypusinated eIF5A and plasmid-based overexpression of a non-hypusinable eIF5A mutant. In this study, we show that SNS01-T is preferentially taken up by malignant B cells, inhibits tumor growth in multiple animal models of B-cell cancers without damaging normal tissues, and synergizes with the current therapies bortezomib and lenalidomide to inhibit tumor progression. The results collectively demonstrate the potential of SNS01-T as a novel therapeutic for treatment of a diverse range of B-cell malignancies.

Mutation of the LXCXE binding cleft of pRb facilitates transformation by ras in vitro but does not promote tumorigenesis in vivo.

BACKGROUND: The Retinoblastoma protein (pRB) is a key tumor suppressor that is functionally inactivated in most cancers. pRB regulates the cell division cycle and cell cycle exit through protein-protein interactions mediated by its multiple binding interfaces. The LXCXE binding cleft region of pRB mediates interactions with cellular proteins that have chromatin regulatory functions. Chromatin regulation mediated by pRB is required for a stress responsive cell cycle arrest, including oncogene induced senescence. The in vivo role of chromatin regulation by pRB during senescence, and its relevance to cancer is not clear. METHODOLOGY/PRINCIPAL FINDINGS: Using gene-targeted mice, uniquely defective for pRB mediated chromatin regulation, we investigated its role during transformation and tumor progression in response to activation of oncogenic ras. We report that the pRB(∆L) mutation confers susceptibility to escape from HrasV12 induced senescence and allows transformation in vitro, although these cells possess high levels of DNA damage. Intriguingly, LSL-Kras, Rb1 (∆L/∆L) mice show delayed lung tumor formation compared to controls. This is likely due to the increased apoptosis seen in the early hyperplastic lesions shortly following ras activation that inhibits tumor progression. Furthermore, DMBA treatment to induce sporadic ras mutations in other tissues also failed to reveal greater susceptibility to cancer in Rb1 (∆L/∆L) mice. CONCLUSIONS/SIGNIFICANCE: Our data suggests that chromatin regulation by pRB can function to limit proliferation, but its loss fails to contribute to cancer susceptibility in ras driven tumor models because of elevated levels of DNA damage and apoptosis.

A context-specific role for retinoblastoma protein-dependent negative growth control in suppressing mammary tumorigenesis.

BACKGROUND: The ability to respond to anti-growth signals is critical to maintain tissue homeostasis and loss of this negative growth control safeguard is considered a hallmark of cancer. Negative growth regulation generally occurs during the G0/G1 phase of the cell cycle, yet the redundancy and complexity among components of this regulatory network has made it difficult to discern how negative growth cues protect cells from aberrant proliferation. METHODOLOGY/PRINCIPAL FINDINGS: The retinoblastoma protein (pRB) acts as the final barrier to prevent cells from entering into the cell cycle. By introducing subtle changes in the endogenous mouse Rb1 gene (Rb1(ΔL)), we have previously shown that interactions at the LXCXE binding cleft are necessary for the proper response to anti-growth signals such as DNA damage and TGF-ß, with minimal effects on overall development. This disrupts the balance of pro- and anti-growth signals in mammary epithelium of Rb1(ΔL/ΔL) mice. Here we show that Rb1(ΔL/ΔL) mice are more prone to mammary tumors in the Wap-p53(R172H) transgenic background indicating that negative growth regulation is important for tumor suppression in these mice. In contrast, the same defect in anti-growth control has no impact on Neu-induced mammary tumorigenesis. CONCLUSIONS/SIGNIFICANCE: Our work demonstrates that negative growth control by pRB acts as a crucial barrier against oncogenic transformation. Strikingly, our data also reveals that this tumor suppressive effect is context-dependent.

Mitotic chromosome condensation mediated by the retinoblastoma protein is tumor-suppressive.

Condensation and segregation of mitotic chromosomes is a critical process for cellular propagation, and, in mammals, mitotic errors can contribute to the pathogenesis of cancer. In this report, we demonstrate that the retinoblastoma protein (pRB), a well-known regulator of progression through the G1 phase of the cell cycle, plays a critical role in mitotic chromosome condensation that is independent of G1-to-S-phase regulation. Using gene targeted mutant mice, we studied this aspect of pRB function in isolation, and demonstrate that it is an essential part of pRB-mediated tumor suppression. Cancer-prone Trp53(-/-) mice succumb to more aggressive forms of cancer when pRB's ability to condense chromosomes is compromised. Furthermore, we demonstrate that defective mitotic chromosome structure caused by mutant pRB accelerates loss of heterozygosity, leading to earlier tumor formation in Trp53(+/-) mice. These data reveal a new mechanism of tumor suppression, facilitated by pRB, in which genome stability is maintained by proper condensation of mitotic chromosomes.

A cancer derived mutation in the retinoblastoma gene with a distinct defect for LXCXE dependent interactions.

BACKGROUND: The interaction between viral oncoproteins such as Simian virus 40 TAg, adenovirus E1A, and human papilloma virus E7, and the retinoblastoma protein (pRB) occurs through a well characterized peptide sequence, LXCXE, on the viral protein and a well conserved groove in the pocket domain of pRB. Cellular proteins, such as histone deacetylases, also use this mechanism to interact with the retinoblastoma protein to repress transcription at cell cycle regulated genes. For these reasons this region of the pRB pocket domain is thought to play a critical role in growth suppression. RESULTS: In this study, we identify and characterize a tumor derived allele of the retinoblastoma gene (RB1) that possesses a discrete defect in its ability to interact with LXCXE motif containing proteins that compromises proliferative control. To assess the frequency of similar mutations in the RB1 gene in human cancer, we screened blood and tumor samples for similar alleles. We screened almost 700 samples and did not detect additional mutations, indicating that this class of mutation is rare. CONCLUSIONS: Our work provides proof of principal that alleles encoding distinct, partial loss of function mutations in the retinoblastoma gene that specifically lose LXCXE dependent interactions, are found in human cancer.

A G1 checkpoint mediated by the retinoblastoma protein that is dispensable in terminal differentiation but essential for senescence.

Terminally differentiated cell types are needed to live and function in a postmitotic state for a lifetime. Cellular senescence is another type of permanent arrest that blocks the proliferation of cells in response to genotoxic stress. Here we show that the retinoblastoma protein (pRB) uses a mechanism to block DNA replication in senescence that is distinct from its role in permanent cell cycle exit associated with terminal differentiation. Our work demonstrates that a subtle mutation in pRB that cripples its ability to interact with chromatin regulators impairs heterochromatinization and repression of E2F-responsive promoters during senescence. In contrast, terminally differentiated nerve and muscle cells bearing the same mutation fully exit the cell cycle and block E2F-responsive gene expression by a different mechanism. Remarkably, this reveals that pRB recruits chromatin regulators primarily to engage a stress-responsive G(1) arrest program.

A functional connection between pRB and transforming growth factor beta in growth inhibition and mammary gland development.

Transforming growth factor beta (TGF-beta) is a crucial mediator of breast development, and loss of TGF-beta-induced growth arrest is a hallmark of breast cancer. TGF-beta has been shown to inhibit cyclin-dependent kinase (CDK) activity, which leads to the accumulation of hypophosphorylated pRB. However, unlike other components of TGF-beta cytostatic signaling, pRB is thought to be dispensable for mammary development. Using gene-targeted mice carrying subtle missense changes in pRB (Rb1(DeltaL) and Rb1(NF)), we have discovered that pRB plays a critical role in mammary gland development. In particular, Rb1 mutant female mice have hyperplastic mammary epithelium and defects in nursing due to insensitivity to TGF-beta growth inhibition. In contrast with previous studies that highlighted the inhibition of cyclin/CDK activity by TGF-beta signaling, our experiments revealed that active transcriptional repression of E2F target genes by pRB downstream of CDKs is also a key component of TGF-beta cytostatic signaling. Taken together, our work demonstrates a unique functional connection between pRB and TGF-beta in growth control and mammary gland development.

The retinoblastoma protein regulates pericentric heterochromatin.

The retinoblastoma protein (pRb) has been proposed to regulate cell cycle progression in part through its ability to interact with enzymes that modify histone tails and create a repressed chromatin structure. We created a mutation in the murine Rb1 gene that disrupted pRb's ability to interact with these enzymes to determine if it affected cell cycle control. Here, we show that loss of this interaction slows progression through mitosis and causes aneuploidy. Our experiments reveal that while the LXCXE binding site mutation does not disrupt pRb's interaction with the Suv4-20h histone methyltransferases, it dramatically reduces H4-K20 trimethylation in pericentric heterochromatin. Disruption of heterochromatin structure in this chromosomal region leads to centromere fusions, chromosome missegregation, and genomic instability. These results demonstrate the surprising finding that pRb uses the LXCXE binding cleft to control chromatin structure for the regulation of events beyond the G(1)-to-S-phase transition.