**7. Genomic impacts of cationic lipids**

To date, cationic lipids have been the most widely used delivery system for delivery of nucleic acids both *in vitro* and *in vivo*. For example, Lipofectin™ is the 1:1 mixture of DOTMA and DOPE. It is the first cationic lipid formulation that was received widespread attention. We found that cationic liposomes such as LF and OF, at concentrations routinely used to obtain efficient delivery of gene based medicines, were able to induce gene expression changes in human epithelial A431 cells (Table 1). Such alterations in gene expressions appeared to be largely dependent upon the physicochemical characteristics of the lipid, wherein OF elicited greater gene expression than LF, i.e., up to 16% of the genes studied (Omidi et al., 2003). We speculate that the surface charge may play a key role in terms of such genotoxicity. In these cells, we witnessed that the affected genes were functionally involved in various cellular processes such as cell proliferation, differentiation and apoptosis. The upregulated or downregulated genes include some important genes such as bcl-2-related protein a1 (BCL2A1), caspase 8 isoform c (CASP8), heat shock protein 70 (HSP70) and 60 (HSP60), annexin a2 (ANXA2), and tubulin beta 5 (TUBB5) (Omidi et al., 2003). Up regulation of caspase-8 clearly impart activation of procaspases and caspases that may provoke activity of a series of apoptotic signaling cascades such as electron carrier protein cytochrome C, adaptor protein Apaf-1, Bcl-2 family, p53 and various transcription factors (Kanduc et al., 2002). Given that the heat shock protein 70 acts as an inhibitor of apoptosis (Li et al., 2000), it's upregulation by OF in A431 cells is deemed to be a cellular compensatory or defense response. We assume that cells recognize the xenobiotics upon their biological properties. To examine such concept, we compared OF genotoxicities within two epithelial cell lines (i.e., A431 and A549 cells).

In A549 cells, the genomic impacts were intriguingly dissimilar compared to that of A431 cells (Table 1). Further, we observed some commonalities in gene expression modulation between two different cell lines (Omidi et al., 2008). Upon EASE analyses, the changes in gene expression fell into a number of various functional genomic ontologies. For example,

Toxicogenomics of Nonviral Cationic Gene Delivery Nanosystems 561

NM\_002026 Fibronectin 1, isoform 1 preproprotein; FN1 ─ NC NC NM\_001238 Cyclin e1, isoform 1; CCNE1 NC NC NC NM\_002186 Interleukin 9 receptor; IL9R NC NC + NM\_002945 Replicationprotein a1 (70kd); RPA1 NC NC NC NM\_003875 Guanine monophosphate synthetase; GMPS ─ NC NC NM\_000887 Integrinalpha x precursor; ITGAX NC NC + NM\_000075 Cyclin-dependent kinase 4, isoform 1; CDK4 NC NC ─

NM\_000970 Ribosomal protein l6; RPL6 ─ NC NC NM\_005319 H1 histone family, member 2; H1F2 NC NC NC NM\_002592 Proliferating cell nuclear antigen; PCNA ─ NC NC NM\_020300 Microsomal glutathione s-transferase 1; MGST1 NC NC NC NM\_021065 H2a histone family, member g; H2AFG NC ─ NC NM\_004832 Glutathione-s-transferase like; GSTTLP28 NC ─ NC NM\_006087 Tubulin, beta, 5; TUBB5 NC ─ NC

NM\_005566 Ldha NC ─ NC NM\_015129 Septin 6; SEP6 NC ─ ─ NM\_004039 Annexin a2; ANXA2 NC ─ NC Table 1. Gene expression changes induced by cationic liposomes in A431 and A549 cells. LF: Lipofectin™ ; OF: Oligofectamine™; NC: no changes; +: upregulation; ─: downregulation;

For example, among the genes upregulated by OF in A549 cells (but not A431 cells), the IL9R gene encodes IL9 receptor protein which is a cytokine receptor that specifically mediates the biological effects of IL9. The ligand binding of this receptor leads to the activation of various JAK kinases and STAT proteins, which connect to different biologic responses, in particular some genetic studies, suggested an association of this gene with the

The heat shock proteins 60 and 70 as well as c-src tyrosine kinase (CSK) were observed to be upregulated in both cell lines (Table 1). Of these, the heat shock proteins family of molecular chaperones appears to act in protein folding, translocation, and assembly into complexes; while CSK is mainly involved in protein-tyrosine kinase activity as well as protein metabolism and modifications. Once looked at the overlapped activities of these genes, we found that they are cooperating mostly to activate the binding activity - we speculate that these genes somehow are collaborating perhaps in terms of protein folding and binding. Since liposomal formulations are being explored for pulmonary drug/gene delivery, and thus their ability to activate IL9R should be assessed when used clinically for lung gene

ASAH

NM\_032959 Dna directed rna polymerase ii polypeptide j, isoform

NM\_002789 Proteasome (prosome, macropain) subunit, alpha

adapted with permission (Barar et al., 2009).

development of asthma (Gaga et al., 2007).

**Gene description LF-**

**A431** 

b; POLR2J NC NC +

type, 4; PSMA4 NC ─ ─

**OF-A431** 

**OF-A549** 

**Gene ID (Accession No.)** 

the upregulated genes by OF nanoliposomes included the genes involved in apoptosis, oxidative stress and external/biotic stimulus (e.g., IL9R, DUSP1, CSK, CSE1L); while the downregulated genes were related to the cell growth and/or cell maintenance, cell proliferation and apoptosis (e.g., SEP6, PSMA4).


the upregulated genes by OF nanoliposomes included the genes involved in apoptosis, oxidative stress and external/biotic stimulus (e.g., IL9R, DUSP1, CSK, CSE1L); while the downregulated genes were related to the cell growth and/or cell maintenance, cell

NM\_004417 Dual specificity phosphatase 1; DUSP1 ─ + + NM\_033356 Caspase 8, isoform c; CASP8 NC + NC

NM\_004049 Bcl2-related protein a1; BCL2A1 NC + NC NM\_003195 Transcription elongation factor a (sii), 2; TCEA2 NC + NC

NM\_000994 Rbosomal protein l32; RPL32 NC + NC NM\_001274 Chk1 checkpoint homolog (s. pombe); CHEK1 NC + NC NM\_002849 Protein tyrosine phosphatase, receptor type, r; PTPRR NC + NC NM\_002156 Heat shock 60kd protein 1 (chaperonin); HSPD1 NC + + NM\_002957 Retinoid x receptor, alpha; RXRA NC + NC NM\_001242 Cd27 antigen; TNFRSF7 NC + NC NM\_006083 Red protein; IK NC + NC L12723 Heat shock protein 70; HSP70 NC + + NM\_004383 C-src tyrosine kinase; CSK NC + +

NM\_005546 Il2-inducible t-cell kinase; ITK NC + NC NM\_006235 Pou domain, class 2, associating factor 1; POU2AF1 NC + NC NM\_002623 Prefoldin 4; PFDN4 NC NC NC NM\_001316 Cse1 chromosome segregation 1-like (yeast); CSE1L NC NC +

NM\_000660 Transforming growth factor, beta 1; TGFB1 NC NC NC NM\_000043 Apoptosis (apo-1) antigen 1; TNFRSF6 NC NC NC NM\_001961 Eukaryotic translation elongation factor 2; EEF2 NC NC NC NM\_001786 Cell division cycle 2 protein, isoform 1; CDC2 NC NC NC NM\_021103 Thymosin beta, TMSB10 NC NC NC NM\_004315 N-acylsphingosineamidohydrolase (acidceramidase); ─ NC NC

Excision repair cross-complementing rodent repair deficiency, complementation group 1 (includes overlapping antisense sequence); ERCC1

**Gene description LF-**

**A431** 

(avian); MYC NC + NC

(alpha, 35kd ); EIF2S1 NC + NC

kinase 3; MAPKAPK3 NC + NC

RPS6KA1 NC NC NC

**OF-A431** 

NC + NC

**OF-A549** 

proliferation and apoptosis (e.g., SEP6, PSMA4).

NM\_002467 V-mycmyelocytomatosis viral oncogene homolog

NM\_004094 Eukaryotic translation initiation factor 2, subunit 1

NM\_004635 Mitogen-activated protein kinase-activated protein

NM\_002953 Ribosomal protein s6 kinase, 90kd, polypeptide 1;

**Gene ID (Accession No.)** 

NM\_001983


Table 1. Gene expression changes induced by cationic liposomes in A431 and A549 cells. LF: Lipofectin™ ; OF: Oligofectamine™; NC: no changes; +: upregulation; ─: downregulation; adapted with permission (Barar et al., 2009).

For example, among the genes upregulated by OF in A549 cells (but not A431 cells), the IL9R gene encodes IL9 receptor protein which is a cytokine receptor that specifically mediates the biological effects of IL9. The ligand binding of this receptor leads to the activation of various JAK kinases and STAT proteins, which connect to different biologic responses, in particular some genetic studies, suggested an association of this gene with the development of asthma (Gaga et al., 2007).

The heat shock proteins 60 and 70 as well as c-src tyrosine kinase (CSK) were observed to be upregulated in both cell lines (Table 1). Of these, the heat shock proteins family of molecular chaperones appears to act in protein folding, translocation, and assembly into complexes; while CSK is mainly involved in protein-tyrosine kinase activity as well as protein metabolism and modifications. Once looked at the overlapped activities of these genes, we found that they are cooperating mostly to activate the binding activity - we speculate that these genes somehow are collaborating perhaps in terms of protein folding and binding.

Since liposomal formulations are being explored for pulmonary drug/gene delivery, and thus their ability to activate IL9R should be assessed when used clinically for lung gene

Toxicogenomics of Nonviral Cationic Gene Delivery Nanosystems 563

NM\_006716 Activator of s phase kinase; ASK NC ─ NC NC NM\_000034 Aldolase a; ALDOα NC NC NC NC NM\_004039 Annexin a2; ANXα2 NC + NC NC NM\_000698 Arachidonate 5-lipoxygenase; ALOX5 NC ─ NC NC NM\_004049 Bcl2-related protein a1; BCL2α1 NC NC NC + NM\_000591 Cd14 antigen precursor; CD14 NC NC + NC NM\_001242 Cd27 antigen; TNFRSF7 NC ─ NC NC

NM\_004383 C-src tyrosine kinase; CSK NC NC NC ─ NM\_003914 Cyclin a1; CCNα1 NC NC + NC NM\_001239 Cyclin h; CCNH NC NC ─ NC

NM\_001801 Cysteine dioxygenase, type i; CDO1 NC NC NC ─

NM\_002186 Interleukin 9 receptor; IL9R NC NC + NC NM\_002358 Mad2-like 1; MAD2L1 NC NC NC +

NM\_000245 Met proto-oncogene precursor; Met + NC ─ NC

NM\_000946 Primase, polypeptide 1 (49kd); PRIM1 NC NC NC +

NM\_002789 Proteasome (prosome, macropain) NC + NC NC

**A431 cells A549 cells** 

DAB1 6:DN A

DAB8 DAB16 DAB16

1; CDC2 NC NC ─ NC

CHEK1 NC NC NC ─

CDK4 NC + NC NC

DUSP1 NC ─ NC +

GMPS NC ─ NC +

H2AFG NC + NC NC

(chaperonin); HSPD1 NC ─ NC NC

factor, eosinophil); IL5 NC NC NC ─

preproprotein; MMP8 NC NC NC ─

(acid ceramidase); ASAH NC NC NC +

beta polypeptide; PCC<sup>β</sup> NC ─ NC NC

1; POU2AF1 NC NC NC ─

PCNA NC NC ─ NC

(fusin); CXCR4 NC NC NC ─

**Description** 

NM\_001786 Cell division cycle 2 protein, isoform

NM\_003467 Chemokine (c-x-c motif), receptor 4

NM\_001274 Chk1 checkpoint homolog (s. pombe);

NM\_000075 Cyclin-dependent kinase 4, isoform 1;

NM\_004417 Dual specificity phosphatase 1;

NM\_021065 H2a histone family, member g;

NM\_002156 Heat shock 60kd protein 1

NM\_002424 Matrix metalloproteinase 8

NM\_004315 N-acylsphingosine amidohydrolase

NM\_006235 Pou domain, class 2, associating factor

NM\_002592 Proliferating cell nuclear antigen;

NM\_000532 Propionyl coenzyme a carboxylase,

NM\_000879 Interleukin 5 (colony-stimulating

NM\_003875 Guanine monophosphate synthetase;

**Gene ID (Accession No.)** 

therapy. The CSK along with some other genes were upregulated in A549 cells treated with cationic lipids similar to what we observed previously in A431 cells (Omidi et al., 2003) and is mainly involved in cell growth and/or cell maintenance. The SEP6 and PSMA4 were downregulated genes by OF in both cell lines. The SEP6 gene is a member of the septin family of GTPases. Members of this family are required for cytokinesis. One version of pediatric acute myeloid leukemia is the result of a reciprocal translocation between chromosomes 11 and X, with the breakpoint associated with the genes encoding the mixedlineage leukemia and septin 2 proteins. This gene encodes four transcript variants encoding three distinct isoforms. An additional transcript variant has been identified, but its biological validity has not been determined. The PSMA4 is a multicatalytic proteinase complex with a highly ordered ring-shaped 20S core structure. They are distributed throughout eukaryotic cells at a high concentration and cleave peptides in an ATP/ubiquitin-dependent process in a non-lysosomal pathway.

Because of the gene expression commonalities and distinctions between the two cell lines, we conceptualized that these cells may respond to the cationic lipid "OF" differently upon their cellular characteristics. These cells appeared to undergo somewhat adaptation upon exposure to xenobiotics, as a result of which they could dynamically respond as expressing/activating related cellular elements for recognition and internalization of the cationic lipid. Of interest, we found that the genotoxicity elicited by the cationic lipid nanosystems were largely dependent upon the structural architecture and/or physicochemical properties of the cationic lipid since no extensive overlap was observed in the gene expression profile induced by either LF or OF in A431 cells. Besides, the responsiveness of the target cells to the lipids could be different since the transfection efficiency is significantly depended upon the target cells and lipids used. Likewise, Filion and Phillips (1997) reported high toxicity rate elicited by some cationic lipids in phagocytic cells such as macrophages and U937 cells, but not in non-phagocytic T lymphocytes.

Taken all these findings together, it seems that for attaining detailed characterization of the toxicogenomics of these lipid delivery systems (based on their molecular structure), the gene expression patterns/profiles need to be determined in different cell types perhaps with known cell surface architecture.
